COMPOSITIONS AND METHODS FOR TREATING CANCER
A small molecule compound IODVA1 has been found to have cellular inhibitory activity against several transformed cell lines including Ras-driven cells. IODVA1 decreases cell-cell and cell-extra cellular matrix interactions and reduces growth of Ras-driven tumors. Applicants also synthesized compound NIRA2 and showed in vitro and in vivo efficacy and potency against models of Ph+(BCR-ABL1) B-ALL and of colon adenocarcinoma xenografts.
The present application hereby claims the benefit of the provisional patent application of the same title, Ser. No. 62/842,839, filed on May 3, 2019, the disclosure of which is herein incorporated by reference in its entirety.
STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCHThis invention was made with government support under CA115611 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUNDCancer is a serious public health problem in the United States and other developed countries. Currently, one in four deaths in the United States is due to cancer. However, many cancers are not responsive to existing treatments, or are only minimally responsive, such that existing therapies are not effective. In addition, appearance of resistance mechanisms to current therapies and relapse continue to be a major impediment in the clinic. The leading therapies for cancer are currently surgery, radiation, targeted and immunotherapies, and chemotherapy. Chemotherapeutic approaches such as antitumor antibiotics, alkylating agents, nitrosourea compounds, vinca alkaloids, steroid hormones, and anti-metabolites form the bulk of therapies available to oncologists. They have undesirable side effects because they don't distinguish between healthy and cancerous tissue. Despite advances in the field of cancer treatment, cancer remains a major health problem.
Thus, there is an urgent need in the art for compositions and methods for treatment of cancer. The present disclosure seeks to address this need in the art.
This application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.
The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.
As used herein, the term “effective amount” means the amount of one or more active components that is sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
The terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to an animal that is the object of treatment, observation and/or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. In some embodiments, the terms refer to humans. In further embodiments, the terms may refer to children.
The terms “pharmaceutically acceptable salts” or “a pharmaceutically acceptable salt thereof” refer to salts prepared from pharmaceutically acceptable, non-toxic acids or bases. Suitable pharmaceutically acceptable salts include metallic salts, e.g., salts of aluminum, zinc, alkali metal salts such as lithium, sodium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts; organic salts; salts of free acids and bases; inorganic salts, e.g., sulfate, hydrochloride, and hydrobromide; and other salts which are currently in widespread pharmaceutical use and are listed in sources well known to those of skill in the art, such as The Merck Index. Any suitable constituent can be selected to make a salt of an active drug discussed herein, provided that it is non-toxic and does not substantially interfere with the desired activity. In addition to salts, pharmaceutically acceptable precursors and derivatives of the compounds can be employed. Pharmaceutically acceptable amides, lower alkyl esters, and protected derivatives of the disclosed actives can also be suitable for use in the compositions and methods disclosed herein. A salt of a compound of this disclosure may be formed between an acid and a basic group of the compound, such as an amino functional group, or a base and an acidic group of the compound, such as a carboxyl functional group. According to another embodiment, the compound is a pharmaceutically acceptable acid addition salt. Acids commonly employed to form pharmaceutically acceptable salts include inorganic acids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, as well as organic acids such as para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, as well as related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and other salts. In one embodiment, pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and especially those formed with organic acids such as maleic acid.
The terms “treat,” “treating” or “treatment,” as used herein, refers to methods of alleviating, abating or ameliorating a disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition either prophylactically and/or therapeutically.
The term “carrier” applied to pharmaceutical compositions of the disclosure refers to a diluent, excipient, or vehicle with which an active compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” (any edition).
The term “compound,” as used herein, is also intended to include any salts, solvates, or hydrates thereof.
The term “alkyl” includes straight, branched chain, or cyclic alkyl groups, such as, but not limited to, methyl, ethyl, propyl, butyl, trifluoromethyl, and tetradecyl.
The term “alkoxy” includes straight, branched chain, or cyclic alkoxy groups, such as, but not limited to, methoxy, ethoxy, propoxy, butoxy, 2-methoxyethoxy, sec-butoxy, hexyloxy, and 2-ethylhexyloxy, tetradecyloxy groups.
The term “aryl” encompasses monocyclic and polycyclic aryl groups which contain only carbons on the first ring. The term “monocyclic aryl” refers to phenyl (where the ring only contains carbons), and the term “polycyclic aryl” refers to napthyl and anthracenyl, to phenyl rings having at least a second ring fused thereto, and to napthyl rings having a third ring fused thereto. In the case of a polycyclic aryl consisting of a phenyl ring having a second or third ring fused thereto, or a napthyl ring having a third ring fused thereto, the additional rings may be aromatic or non-aromatic carbocyclic or heterocyclic rings, provided that in such cases the point of attachment will be to the carbocyclic aromatic ring. For example, a subset of this aryl group is a polycyclic aryl group wherein the second ring is a “heteroaryl” which contains carbon atoms and at least one heteroatom selected from the group consisting of O, N, and S (provided that O and S cannot be adjacent to each other in the same ring). Alternatively, a ring carbon atom of the second and/or third further rings may be replaced with a carbonyl [—C(═O) group] (e.g., when such rings are non-aromatic). “Substituted aryl” refers to an aryl group substituted by one or more substituents, preferably 1 to 4 substituents (more preferably 1 or 2), at any point of attachment of any ring, selected from alkyl, substituted alkyl, and the substituents recited above for substituted alkyl groups.
Accordingly, examples of aryl groups that are of interest in forming compounds of the invention include:
and, additionally, similar structures.
The terms “heterocycle”, “heterocyclic” and “heterocyclo” refer to fully saturated, partially unsaturated, or fully unsaturated, including aromatic (i.e., “heteroaryl”) cyclic groups (for example, 3 to 7 membered monocyclic, 7 to 11 membered bicyclic, or 10 to 16 membered tricyclic ring systems) which have at least one heteroatom in at least one carbon atom-containing ring. Thus, the term “heteroaryl” is a subset of heterocyclo groups. Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3, or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized, provided sulfur and oxygen are not adjacent to each other in the ring. (The term “heteroarylium” refers to a heteroaryl group bearing a quaternary nitrogen atom and thus a positive charge.) Additionally, one or more (preferably one) carbon ring atoms of the heterocyclo ring may, as valence allows, be replaced with carbonyl group, i.e., —C(═O)—. The heterocyclic group may be attached to the remainder of the molecule at any heteroatom or carbon atom of the ring or ring system.
Exemplary monocyclic heterocyclic groups include those selected from the group consisting of ethylene oxide, azetidinyl, pyrrolidinyl, pyrrolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, hexahydrodiazepinyl, 4-piperidonyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, triazolyl, tetrazolyl, tetrahydropyranyl, morpholinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, 1,3-dioxolane and tetrahydro-1,1-dioxothienyl, and the like.
Exemplary bicyclic heterocyclic groups include those selected from the group consisting of indolyl, isoindolyl, benzothiazolyl, benzodioxolyl, benzoxazolyl, benzoxadiazolyl, benzothienyl, quinuclidinyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, benzofurazanyl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,2-b]pyridinyl] or furo[2,3-b]pyridinyl), dihydrobenzodioxinyl, dihydrodioxidobenzothiophenyl, dihydroisoindolyl, dihydroindolyl, dihydroquinolinyl, dihydroquinazolinyl (such as 3,4-dihydro-4-oxo-quinazolinyl), triazinylazepinyl, tetrahydroquinolinyl and the like. Exemplary tricyclic heterocyclic groups include carbazolyl, benzidolyl, phenanthrolinyl, dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyl and the like.
The term “heterocyclene” refers to bivalent heterocycle groups as defined above.
“Substituted heterocycle”, “substituted heterocyclic” and “substituted heterocyclo” (such as “substituted heteroaryl”) refer to heterocycle, heterocyclic or heterocyclo groups substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment, wherein the substituents arc selected from those recited above for substituted cycloalkyl groups.
The term “group” is used, it is intended to encompass not only the substituent's unsubstituted form, but also its form further substituted with any substituent group or groups as herein mentioned, so long as the substituent does not destroy properties necessary for utility. Suitably, a substituent group may be halogen or may be bonded to the remainder of the molecule by an atom of carbon, nitrogen, oxygen, or sulfur.
In one aspect, a composition that may be used according to the disclosed methods may comprise a compound having the following structure:
(referred to herein as “Compound 1,” or “an IODVA1 compound”) and a pharmaceutically acceptable carrier;
-
- wherein A=NH, NRB, S, O, C═C, N═C, C═N
- wherein R1, R2 are independently substituted or unsubstituted aryl or heteroaryl rings
- wherein R3=singly or multiply substituted as H, D, Halo, CN, C1-C4 Alkyl, C1-C4 Alkoxy, C1-C4 Alkylsulfonyl, C1-C4 Alkyl amino, or C1-C4 mercapto
- wherein R8=H, M;
- and all tautomers thereof.
In one aspect, the compound may have the structure
-
- wherein A=NH, S
- wherein R1, R2 are independently substituted or unsubstituted phenyl, pyridyl, furanyl, pyrimidinyl, triazinyl, or diazinyl rings
- wherein R3=singly or multiply substituted as H, D, Halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et, or Pr;
- and all tautomers thereof.
In one aspect, the compound may have the structure
-
- and wherein R1, R2 are independently substituted or unsubstituted phenyl, pyridyl, furanyl, pyrimidinyl rings
- wherein R3=singly or multiply substituted as H, D, Halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et, or Pr,
- and all tautomers thereof.
In one aspect, the compound may have the structure
-
- wherein R4-R17 are independently selected from H, D, Halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et, or Pr,
- and all tautomers thereof.
In one aspect, the compound may have the structure
-
- wherein R4-R17 are independently selected from H, D, Halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et, or Pr,
- and all tautomers thereof.
In one aspect, the compound may have the structure
-
- wherein R4-R7 are independently selected from H, D, F, Cl, CN, OH, OMe, SMe, Me, or Et;
- wherein R9-R17 are independently selected from H, D, F, Cl, CN, OH, OMe, SO2Me, NHMe, NMe2, Me, or Et;
- and all tautomers thereof.
In one aspect, the compound may have the structure
-
- wherein R4-R7 are independently selected from H, D, F, Cl, CN, OH, OMe, SMe, Me, or Et;
- wherein R9-R17 are independently selected from H, D, F, Cl, CN, OH, OMe, SO2Me, NHMe, NMe2, Me, or Et,
- and all tautomers thereof.
In one aspect, the compound may have the structure
-
- wherein R4-R7 are independently selected from H, D, F, OH, OMe, Me;
- wherein R9-R17 are independently selected from H, D, F, Cl, CN, OH, OMe, SO2Me, NHMe, NMe2, Me, or Et,
- and all tautomers thereof.
In one aspect, the compound may have the structure
-
- wherein R5, R6 are independently selected from H, D, F, Cl, OH, OMe, or Me;
- wherein R9-R17 are independently selected from H, D, F, Cl, OH, OMe, or Me;
- wherein each ring bears <2 non-H substituents;
- and all tautomers thereof.
In one aspect, the compound may have the structure
and all tautomers thereof.
In one aspect, the compound may have the structure
-
- and all tautomers thereof.
In one aspect, a composition comprising a compound having the structure
and a pharmaceutically acceptable carrier is disclosed.
In one aspect, the compound may have the structure
wherein A is selected from NH, NRB, S, O, C═C, N═C, C═N; wherein R1, R2 are independently substituted or unsubstituted* aryl or heteroaryl rings; wherein R3 is singly or multiply substituted as H, D, Halo, CN, C1-C4 Alkyl, C1-C4 Alkoxy, C1-C4 Alkylsulfonyl, C1-C4 Alkyl amino, or C1-C4 mercapto; wherein R8 is H or Me; and all tautomers thereof.
In one aspect, the compound may have the structure
wherein A is NH or S; wherein R1, R2 are independently substituted or unsubstituted phenyl, pyridyl, furanyl, pyrimidinyl, triazinyl, or diazinyl rings; wherein R3 is singly or multiply substituted as H, D, Halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et, or Pr; and all tautomers thereof.
In one aspect, the compound may have the structure
wherein R1, R2 are independently substituted or unsubstituted phenyl, pyridyl, furanyl, pyrimidinyl rings; wherein R3 is singly or multiply substituted as H, D, Halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et, or Pr; and all tautomers thereof.
In one aspect, the compound may have the structure
wherein R4-R17 are each independently selected from H, D, Halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et, or Pr; and all tautomers thereof.
In one aspect, the compound may have the structure
wherein R4-R17 are each independently selected from H, D, Halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et, or P; and all tautomers thereof.
In one aspect, the compound may have the structure
wherein R4-R7 are each independently selected from H, D, F, Cl, CN, OH, OMe, SMe, Me, or Et; wherein R9-R17 are each independently selected from H, D, F, Cl, CN, OH, OMe, SO2Me, NHMe, NMe2, Me, or Et; and all tautomers thereof.
In one aspect, the compound may have the structure
wherein R4-R7=H, D, F, Cl, CN, OH, OMe, SMe, Me, or Et; wherein R9-R17=H, D, F, Cl, CN, OH, OMe, SO2Me, NHMe, NMe2, Me, or Et, and all tautomers thereof.
In one aspect, the compound may have the structure
wherein R4-R7=Independently H, D, F, OH, OMe, Me; wherein R9-R17 may be independently selected from H, D, F, Cl, CN, OH, OMe, SO2Me, NHMe, NMe2, Me, or Et, and all tautomers thereof.
In one aspect, the compound may have the structure
wherein R4-R7=Independently H, D, F, Cl, OH, OMe, or Me; wherein R9-R12 and R14-R17 may be independently selected from H, D, F, Cl, OH, OMe, or Me. Each ring bearing ≤2 non-H substituents; and all tautomers thereof.
In one aspect, a composition comprising
and all tautomers thereof, and a pharmaceutically acceptable carrier is disclosed.
In one aspect, a composition comprising
and all tautomers thereof, and a pharmaceutically acceptable carrier, is disclosed.
In one aspect, composition comprising what is herein referred to as the NIRA2 class of compounds, is disclosed. The composition may comprise a compound having the structure:
wherein R1, R2, and R4 are independently selected from H, D, Halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et, or Pr, and all tautomers thereof, wherein R3 is singly or multiply substituted as H, D, Halo, CN, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylsufonyl; C1-C4 alkylamino, or C1-C4 mercapto; wherein A is selected from NH, NRB, S, O, C═C, N═C, C═N, wherein R8 is H or Me, wherein each Q is independently selected from N, C, and S; and a pharmaceutically acceptable carrier.
In one aspect, the compound may have the structure
wherein R5-R20 are independently selected from H, D, Halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NMe2, Me, Et, or Pr, wherein each Q is independently selected from N, C, and S, and all tautomers thereof; and a pharmaceutically acceptable carrier.
In one aspect, the compound may have the structure
wherein R1, R2, and R4 are independently selected from H, D, Halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et, or Pr, and all tautomers thereof;
wherein R3 is singly or multiply substituted as H, D, Halo, CN, C1-C4 alkyl; C1-C4 alkoxy; C1-C4 alkysulfonyl; C1-C4 akylamino; or C1-C4 mercapto; wherein A is selected from NH, NR8, S, O, C═C, N═C, C═N, wherein R8 is H or Me, wherein each Q is independently selected from N, C, and S; and a pharmaceutically acceptable carrier.
In one aspect, the compound may have the structure
or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In one aspect, the compound may have the structure
or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
In one aspect, any of the aforementioned compositions may be free of, or substantially free of one or both of the following compounds:
In one aspect, a composition comprising
and a pharmaceutically acceptable carrier is disclosed.
In one aspect, a method of treating cancer in an individual in need thereof, is disclosed. The method may comprise the step of administering to said individual a compound or composition as disclosed herein.
In one aspect, the cancer may be a solid tumor. The treating step may effect a decrease in tumor volume.
In one aspect, the treating step may effect a decrease in cancer cell proliferation. In one aspect, the treating step may affect an increase in cancer cell death.
In one aspect, the cancer may be selected from a leukemia, preferably ALL, AML, or MLL, chemotherapy-resistant leukemia, immunotherapy-resistant leukemia, relapsed leukemia, and other targeted-therapy resistant leukemias. In one aspect, the cancer may be selected from adenocarcinoma, breast cancer. In one aspect, the cancer may be one in which Vav3 is overexpressed, such as prostate cancer, ovarian cancer, endometrial cancer, thyroid cancer, lung cancer, particularly non-small cell lung cancer, colorectal cancer, pancreatic cancer, and cervical cancer. In one aspect, the cancer may be a Ras-driven cancer, including RASopathies, for example, NF1 or MPNST. In one aspect, the cancer is any cancer that overexpresses Vav3.
In a further aspect, a method of treating a cancer based on the status of Vav3 expression is disclosed. In this aspect, the method may comprise the step of determining the level of Vav3 in a biopsy obtained from a cancer in an individual; and administering a composition as described herein to said individual where said level of Vav3 is elevated as compared to a control.
Pharmaceutical Compositions
The compositions may be administered in oral dosage forms such as tablets, capsules (each of which includes sustained release or timed-release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. They may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, intralesional, or intramuscular forms all utilizing dosage forms well known to those of ordinary skill in the pharmaceutical arts. The compositions may be administered by intranasal route via topical use of suitable intranasal vehicles, or via a transdermal route, for example using conventional transdermal skin patches. A dosage protocol for administration using a transdermal delivery system may be continuous rather than intermittent throughout the dosage regimen.
In one aspect, pharmaceutical compositions are isotonic with the blood or other body fluid of the recipient. The isotonicity of the compositions may be attained using sodium tartrate, propylene glycol or other inorganic or organic solutes. An example includes sodium chloride. Buffering agents may be employed, such as acetic acid and salts, citric acid and salts, boric acid and salts, and phosphoric acid and salts. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
Viscosity of the pharmaceutical compositions may be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is useful because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. In some embodiments, the concentration of the thickener will depend upon the thickening agent selected. An amount may be used that will achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents.
A pharmaceutically acceptable preservative may be employed to increase the shelf life of the pharmaceutical compositions. Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed. A suitable concentration of the preservative is typically from about 0.02% to about 2% based on the total weight of the composition, although larger or smaller amounts may be desirable depending upon the agent selected. Reducing agents, as described above, may be advantageously used to maintain good shelf life of the formulation.
In one aspect, active agents provided herein may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, or the like, and may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. See, e.g., “Remington: The Science and Practice of Pharmacy”, Lippincott Williams & Wilkins; 20th edition (Jun. 1, 2003) and “Remington's Pharmaceutical Sciences,” Mack Pub. Co.; 18th and 19th editions (December 1985, and June 1990, respectively). Such preparations may include complexing agents, metal ions, polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, and the like, liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. The presence of such additional components may influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application, such that the characteristics of the carrier are tailored to the selected route of administration.
For oral administration, the pharmaceutical compositions may be provided as a tablet, aqueous or oil suspension, dispersible powder or granule, emulsion, hard or soft capsule, syrup or elixir. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and may include one or more of the following agents: sweeteners, flavoring agents, coloring agents and preservatives. Aqueous suspensions may contain the active ingredient in admixture with excipients suitable for the manufacture of aqueous suspensions.
Formulations for oral use may also be provided as hard gelatin capsules, wherein the active ingredient(s) are mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules. In soft capsules, the active agents may be dissolved or suspended in suitable liquids, such as water or an oil medium, such as peanut oil, olive oil, fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers and microspheres formulated for oral administration may also be used. Capsules may include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredient in admixture with fillers such as lactose, binders such as starches, and/or lubrimayts such as talc or magnesium stearate and, optionally, stabilizers.
Tablets may be uncoated or coated by known methods to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period of time. For example, a time delay material such as glyceryl monostearate may be used. When administered in solid form, such as tablet form, the solid form typically comprises from about 0.001 wt. % or less to about 50 wt. % or more of active ingredient(s), for example, from about 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 wt. % to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 wt. %.
Tablets may contain the active ingredients in admixture with non-toxic pharmaceutically acceptable excipients including inert materials. For example, a tablet may be prepared by compression or molding, optionally, with one or more additional ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered active agent moistened with an inert liquid diluent.
In some embodiments, each tablet or capsule contains from about 1 mg or less to about 1,000 mg or more of an active agent provided herein, for example, from about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg to about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 900 mg. In some embodiments, tablets or capsules are provided in a range of dosages to permit divided dosages to be administered. A dosage appropriate to the patient and the number of doses to be administered daily may thus be conveniently selected. In certain embodiments two or more of the therapeutic agents may be incorporated to be administered into a single tablet or other dosage form (e.g., in a combination therapy); however, in other embodiments the therapeutic agents may be provided in separate dosage forms.
Suitable inert materials include diluents, such as carbohydrates, mannitol, lactose, anhydrous lactose, cellulose, sucrose, modified dextrans, starch, and the like, or inorganic salts such as calcium triphosphate, calcium phosphate, sodium phosphate, calcium carbonate, sodium carbonate, magnesium carbonate, and sodium chloride. Disintegrants or granulating agents may be included in the formulation, for example, starches such as corn starch, alginic acid, sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite, insoluble cationic exchange resins, powdered gums such as agar, karaya, or alginic acid or salts thereof.
Binders may be used to form a hard tablet. Binders include materials from natural products such as acacia, starch and gelatin, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, and the like.
Lubricants, such as stearic acid or magnesium or calcium salts thereof, polytetrafluoroethylene, liquid paraffin, vegetable oils and waxes, sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol, starch, talc, pyrogenic silica, hydrated silicoaluminate, and the like, may be included in tablet formulations.
Surfactants may also be employed, for example, anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate, cationic such as benzalkonium chloride or benzethonium chloride, or nonionic detergents such as polyoxyethylene hydrogenated castor oil, glycerol monostearate, polysorbates, sucrose fatty acid ester, methyl cellulose, or carboxymethyl cellulose.
Controlled release formulations may be employed wherein the active agent or analog(s) thereof is incorporated into an inert matrix that permits release by either diffusion or leaching mechanisms. Slowly degenerating matrices may also be incorporated into the formulation. Other delivery systems may include timed release, delayed release, or sustained release delivery systems.
Coatings may be used, for example, nonenteric materials such as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols, or enteric materials such as phthalic acid esters. Dyestuffs or pigments may be added for identification or to characterize different combinations of active agent doses.
When administered orally in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added to the active ingredient(s). Physiological saline solution, dextrose, or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol are also suitable liquid carriers. The pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive or arachis oil, a mineral oil such as liquid paraffin, or a mixture thereof. Suitable emulsifying agents include naturally occurring gums such as gum acacia, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsions may also contain sweetening and flavoring agents.
Pulmonary delivery of the active agent may also be employed. The active agent may be delivered to the lungs while inhaling and traverses across the lung epithelial lining to the blood stream. A wide range of mechanical devices designed for pulmonary delivery of therapeutic products may be employed, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. These devices employ formulations suitable for the dispensing of active agent. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants, and/or carriers useful in therapy. The active ingredients may be prepared for pulmonary delivery in particulate form with an average particle size of from 0.1 um or less to 10 um or more, for example, from about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 um to about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5 um. Pharmaceutically acceptable carriers for pulmonary delivery of active agent include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include DPPC, DOPE, DSPC, and DOPC. Natural or synthetic surfactants may be used, including polyethylene glycol and dextrans, such as cyclodextran. Bile salts and other related enhancers, as well as cellulose and cellulose derivatives, and amino acids may also be used. Liposomes, microcapsules, microspheres, inclusion complexes, and other types of carriers may also be employed.
Pharmaceutical formulations suitable for use with a nebulizer, either jet or ultrasonic, typically comprise the active agent dissolved or suspended in water at a concentration of about 0.01 or less to 100 mg or more of active agent per mL of solution, for example, from about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg to about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 mg per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the active agent caused by atomization of the solution in forming the aerosol.
Formulations for use with a metered-dose inhaler device generally comprise a finely divided powder containing the active ingredients suspended in a propellant with the aid of a surfactant. The propellant may include conventional propellants, such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, and hydrocarbons. Example propellants include trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, 1,1,1,2-tetrafluoroethane, and combinations thereof. Suitable surfactants include sorbitan trioleate, soya lecithin, and oleic acid.
Formulations for dispensing from a powder inhaler device typically comprise a finely divided dry powder containing active agent, optionally including a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in an amount that facilitates dispersal of the powder from the device, typically from about 1 wt. % or less to 99 wt. % or more of the formulation, for example, from about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 wt. % to about 55, 60, 65, 70, 75, 80, 85, or 90 wt. % of the formulation.
In some embodiments, an active agent provided herein may be administered by intravenous, parenteral, or other injection, in the form of a pyrogen-free, parenterally acceptable aqueous solution or oleaginous suspension. Suspensions may be formulated according to methods well known in the art using suitable dispersing or wetting agents and suspending agents. The preparation of acceptable aqueous solutions with suitable pH, isotonicity, stability, and the like, is within the skill in the art. In some embodiments, a pharmaceutical composition for injection may include an isotonic vehicle such as 1,3-butanediol, water, isotonic sodium chloride solution, Ringer's solution, dextrose solution, dextrose and sodium chloride solution, lactated Ringer's solution, or other vehicles as are known in the art. In addition, sterile fixed oils may be employed conventionally as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the formation of injectable preparations. The pharmaceutical compositions may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
The duration of the injection may be adjusted depending upon various factors, and may comprise a single injection administered over the course of a few seconds or less, to 0.5, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more of continuous intravenous administration.
Dosage
In one aspect, an agent disclosed herein may be present in an amount of from about 0.5% to about 95%, or from about 1% to about 90%, or from about 2% to about 85%, or from about 3% to about 80%, or from about 4%, about 75%, or from about 5% to about 70%, or from about 6%, about 65%, or from about 7% to about 60%, or from about 8% to about 55%, or from about 9% to about 50%, or from about 10% to about 40%, by weight of the composition.
In one aspect, the compounds may be administered at the rate of 100 μg to 1000 mg per day per kg of body weight. Orally, the compounds may be suitably administered at the rate of about 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg to about 1, 5, 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mg per day per kg of body weight. The required dose can be administered in one or more portions. For oral administration, suitable forms are, for example, tablets, gel, aerosols, pills, dragees, syrups, suspensions, emulsions, solutions, powders and granules; one method of administration includes using a suitable form containing from 1 mg to about 500 mg of active substance. In one aspect, administration may comprise using a suitable form containing from about 1, 2, 5, 10, 25, or 50 mg to about 100, 200, 300, 400, 500 mg of active substance.
A dosage regimen will vary depending upon known factors such as the pharmacodynamic and pharmacokinetic characteristics of the agents and their mode and route of administration; the species, age, sex, health, medical condition, and weight of the patient, the nature and extent of the symptoms, the kind of concurrent treatment, the frequency of treatment, the route of administration, the renal and hepatic function of the patient, and the desired effect. The effective amount of a drug required to prevent, counter, or arrest progression of a symptom or effect of a muscle contracture can be readily determined by an ordinarily skilled physician. In one aspect, active agents provided herein may be administered in a dosage form selected from intravenous or subcutaneous unit dosage form, oral, parenteral, intravenous, and subcutaneous. In some embodiments, active agents provided herein may be formulated into liquid preparations for, e.g., oral administration. Suitable forms include suspensions, syrups, elixirs, and the like. In some embodiments, unit dosage forms for oral administration include tablets and capsules. Unit dosage forms configured for administration once a day; however, in certain embodiments it may be desirable to configure the unit dosage form for administration twice a day, or more.
In some embodiments, active agents provided herein may additionally employ adjunct components conventionally found in pharmaceutical compositions in their art-established fashion and at their art-established levels. Thus, for example, the compositions may contain additional compatible pharmaceutically active materials for combination therapy or may contain materials useful in physically formulating various dosage forms, such as excipients, dyes, thickening agents, stabilizers, preservatives or antioxidants.
In some embodiments, the active agents provided herein may be provided to an administering physician or other health care professional in the form of a kit. The kit is a package which houses a container which contains the active agent(s) in a suitable pharmaceutical composition, and instructions for administering the pharmaceutical composition to a subject. The kit may optionally also contain one or more additional therapeutic agents currently employed for treating a disease state as described herein. For example, a kit containing one or more compositions comprising active agents provided herein in combination with one or more additional active agents may be provided, or separate pharmaceutical compositions containing an active agent as provided herein and additional therapeutic agents may be provided. The kit may also contain separate doses of an active agent provided herein for serial or sequential administration. The kit may optionally contain one or more diagnostic tools and instructions for use. The kit may contain suitable delivery devices, e.g., syringes, and the like, along with instructions for administering the active agent(s) and any other therapeutic agent. The kit may optionally contain instructions for storage, reconstitution (if applicable), and administration of any or all therapeutic agents included. The kits may include a plurality of containers reflecting the number of administrations to be given to a subject.
EXAMPLESThe following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Ras is a target in several human cancers and in a set of genetic diseases termed RASopathies (Aoki et al., 2016; McCormick, 2016; Simanshu et al., 2017; Tidyman and Rauen, 2009b). Applicants targeted the GTP-bound form of the G60A point mutant previously described by the applicants (Ford et al., 2005). Targeting the RasG60A structure by virtual screening was justified as follows. First, a potential binding site cleft situated between the switch 1 and the triphosphate nucleotide, albeit small, can be identified in this structure. This cleft results from the adoption of the switch 1 region in the GTP- but not GDP-bound structure of this mutant. This conformation is different from wild-type Ras, but similar to nucleotide-free Ras in complex with the guanine nucleotide exchange factor Sos (Boriack-Sjodin et al., 1998), which applicants terms ‘the open conformation’ of Ras. Switch 1 corresponds to residues 24-40 and is responsible for GTP-/Mg2+-coordination and binding to effectors and regulators (Pai et al., 1989; Vetter and Wittinghofer, 2001; Wittinghofer and Nassar, 1996). Second, this mutation severely attenuates the binding of Ras to its effector, Raf kinase, in vitro (Ford et al., 2005) and reverts the transforming ability of constitutively active Ras in cells (Hwang et al., 1996; Sung et al., 1995; Sung et al., 1996). Third, it was previously shown using solution 31P-NMR spectroscopy that GTP-bound Ras adopts two conformations in equilibrium (Geyer et al., 1996; Spoerner et al., 2010; Spoerner et al., 2007): one is capable of effector binding and therefore signaling, while the other is non-signaling and is mimicked by the G60A and T35S mutants (Araki et al., 2011; Shima et al., 2010; Spoerner et al., 2001). Taken together, it is believed that a small molecule that keeps Ras in the open conformation may inhibit its signaling. A similar approach has previously led to the discovery of the anti-Ras ‘Kobe’ compounds (Shima et al., 2013).
Combined data from virtual screening, cell growth and colony formation assays, and chemical synthesis and analysis identified a small molecule termed IODVA1. with cellular inhibitory activity against several transformed cell lines including Ras-driven cells. Applicants showed that IODVA1 targets Rac activation and signaling. Applicants have demonstrated that IODVA 1 has in vivo activity against human MDA-MB-231 triple negative breast cancer (TNBC) and H2122 non-small cell lung cancer (NSCLC) cell lines in xenograft mouse tumor models.
Results
Docking.
Using the Autodock program (Huey et al., 2007; Morris et al., 1998; Morris et al., 2009), Applicants performed virtual screening for small molecules that could potentially fit into the identified Ras binding interface pocket. The pocket is lined by Ile21, Gln22, switch 1 residues Gln25-Pro34, Lys147 and Arg149 and the GTP-ribose (
Cellular & Biochemical Phenotypic Screening Assays.
To assess the effect on proliferation, the 40 hit compounds were tested in cellular assays. Hits at 10 μM were screened against the human lung mucoepidermoid carcinoma cell line H292 and the human lung adenocarcinoma cell line A549 using the MTS cell proliferation assay (Berridge et al., 2005; Denizot and Lang, 1986; Loveland et al., 1992). These two epithelial cell lines were selected because H292 encodes wild-type RAS (RASWT) while A549 encodes KRASG12V mutation with the expectation that compounds that differentiate between these two cell lines should be specific for oncogenic vs. RASWT.
While NSC124205 at 5 μM inhibited the growth of yeast strains containing single mutations in the rad50, mec2, and bub3 genes and doubly mutant sgs1+mgt1, cln2+rad14, and mlh1+rad18 strains at better than 80% compared to untreated controls. The results of the NCI screen are consistent with those shown here.
Biochemistry of NSC124205.
Applicants tested the ability of NSC124205 and 7 other compounds to acutely decrease ERK and AKT phosphorylation in 3T3 cells. Cells were serum starved for 24 h, incubated with the DMSO vehicle control or with the compounds at 15 μM for 1 h, EGF stimulated for 5 min, and change in ERK- and AKT-phosphorylation was checked by immunoblotting.
To probe the effect of NSC124205 on oncogenic Ras signaling, Applicants generated NIH-3T3 cells overexpressing wild-type or dominant active HRASG12V (
Inhibition of Anchorage-Independent Growth.
NSC124205 was screened for its ability to inhibit proliferation of the HRASG12V overexpressing 3T3 cells using the soft agar colony formation assay at concentrations of 5 and 25 μM. As shown in
Chemical Synthesis of IODVA1.
Before entering in vivo studies, Applicants checked compound NSC124205's identity and purity. Applicants tested a freshly made DMSO solution of NSC124205 obtained from the NCI by high-performance liquid chromatography-mass spectroscopy (HPLC/MS). As shown in
Applicants focused on the structure shown in
The synthesis is derived from analogous molecules in the literature. 2-guanidinobenzimidazole and α-pyridoin were heated in presence of acetic acid at 90° C. HPLC analysis of the synthesized compound shows that a single band is observed at 12.8 min corresponding to peak 1b observed in the NSC124205 sample (
Analysis of IODVA1 shows that it has some favorable drug-like properties despite the large nitrogen content and aromatic character. Applicants performed several calculations to predict the drugability of IODVA1. Calculations show that the target molecule is non-planar, reasonably soluble in aqueous phase with a log P of 3.25, has one hydrogen-bond donor and four hydrogen-bond acceptors. Because of these chemical properties, IODVA1 follows the Lipinski's rule of 5.
Effects of IODVA1 on Proliferation of Cells Harboring Activated Ras.
Applicants tested IODVA1's ability to inhibit the proliferation of cells harboring activated Ras and therefore its ability to recapitulate the compound obtained from the NCI. Applicants chose the NF1-associated malignant peripheral nerve sheath tumors (MPNST) cell line ST8814, which is characterized by active wild-type Ras (Basu et al., 1992; Mahller et al., 2006) and the triple negative breast cancer cell line MDA-MB-231 carrying the oncogenic KRASG13D mutation (Hollestelle et al., 2007). In addition, applicants used the non-transformed mammary epithelial cell line MCF10A (RASWT immortalized by a spontaneous t(3;9)(3p13;9p22) translocation that deletes the CDKN2A gene, also known as p16) and the non-invasive estrogen receptor (ER) and progesterone receptor (PR) positive RASWT breast cancer cell line MCF7 (wild-type p53) and T47D (mutant p53). Cells were grown in growth media containing IODVA1 at concentrations between 0.1 and 10 μM or vehicle control and cell number was counted using the trypan blue exclusion method.
IODVA1 and Ras Activation.
To check if IODVA1 inhibits Ras activation in cells, applicants determined the levels of active-GTP-bound-Ras in ST88-14 cells treated with IODVA1 (2 μM) or vehicle control at various time points. GST-RafRBD bound glutathione-beads were incubated with ST8814 cell lysates, thoroughly washed, and the protein complex separated on SDS-PAGE. Levels of GTP-bound pan-RAS proteins bound to RafRBD were determined by immunoblotting. As shown in
IODVA1 Interferes with Lamellipodia and Circular Dorsal Ruffle Formation.
Progression of cancer invasion and metastasis requires the aberrant activation of cell migration, which is driven by the reorganization of the actin cytoskeleton (Condeelis et al., 2005; Sahai, 2005; Yamaguchi and Condeelis, 2007; Yamaguchi et al., 2005). A major trait of Ras-transformed cells is a reorganized actin cytoskeleton, which leads to poor adhesion, increased motility, invasiveness, and contact-independent growth. To check the effect of IODVA1 on overall actin cytoskeleton structures, MDA-MB-231 cells were serum starved for 4 h, treated with IODVA1 (0-3 μM) for 1 h, then stimulated cells with EGF for 30 min, which induces lamellipodia formation.
Lamellipodium formation and membrane ruffling in response to growth factor stimulation is characteristic of Rac activation (Ridley and Hall, 1992; Ridley et al., 1992). Based on the observation that IODVA1 treatment impedes formation of lamellipodia, applicants evaluated the action of IODVA1 on another Rac-mediated actin structure—circular dorsal ruffles (CDRs) (Steffen et al., 2013). CDRs are enclosed, dynamic, ring-shaped structures that erect vertically and appear on the dorsal surface of cells and Rac activity is required for their formation. 3T3 fibroblasts were starved for 4 h, treated with IODVA1 (0-3 μM) for 1 h, and stimulated with PDGF for 10 min.
IODVA1 Inhibits Rac-Activation.
To confirm that IODVA1 interferes with Rac activation, applicants checked the levels of active Rac and its downstream effector PAK1/2. MDA-MB-231 cells were incubated with IODVA1 (0-3 μM) for 1 h and levels of GTP-bound active Rac were measured by GST-PAK-GBD pull-downs and quantified (
IODVA1 May Decrease Cell-ECM and Cell-Cell Interactions.
Applicants assessed if IODVA1 interferes with cell spreading on the extracellular matrix, an event that is also governed by the rearrangement of the actin cytoskeleton. MCF7, T47D, or MDA-MB-231 cells were plated on fibronectin coated coverslips for 10 min, incubated for additional 30 min in the presence of 0-3 μM IODVA1. fixed and examined by bright field microscopy.
In vitro 3D assays, such as spheroid formation, serve as an intermediate between 2D (monolayer) cellular assays and in vivo animal models. Spheroid formation is mediated by matrix development and remodeling and changes in the cytoskeleton and cell-cell contacts and adhesion. To evaluate the effect of IODVA1 on spheroid formation, single cell suspension were plated in complete media containing vehicle control or IODVA1 at 0.1-3 μM range using the hanging drop and ultra-low attachment (ULA) methods (Foty R. 2011).
In the hanging drop method, spheroids were mixed with the indicated concentrations of IODVA1 in complete media and spheroids were allowed to form in 25 μL hanging drops on the lid of a 10-cm dish. After 96 h, spheroids were transferred into a 10-cm dish using a wide-barrel pipet tip and imaged before and after trituration using bright field microscopy.
For the spheroid formation in the ultra-low attachment plates, MCF7, T47D, and MDA-MB-231 (5,000 cells) were incubated in complete media with IODVA1 at 0-3 μM for 5 days. Aggregates and spheroids were dissociated with Accutase and live cells were counted using trypan blue exclusion. Treatment with IODVA1 significantly decreased the number of live cells in a spheroid (
IODVA1 Kinase Inhibitory Activity.
IODVA1 has two pyridine groups attached to a central imidazole group. Pyridine is among the most common scaffolds found in kinase inhibitors (Xing et al., 2014) and is found in several potent inhibitors of FLT3, Aurora, ROCK, AKT, and other kinases (Bavetsias et al., 2012; Green et al., 2015; Woods et al., 2006) suggesting that IODVA1 might have kinase inhibitory activity. To test this hypothesis, applicants evaluated the potential of IODVA1 to interfere with the ability of 369 recombinant wild-type kinases to hydrolyze ATP. Each kinase was tested twice at one single IODVA1 concentration of 0.5 μM and data were averaged and compared to vehicle DMSO control. A plot of the replicates compared to vehicle control set at 100% is shown in
IODVA1 Reduces Tumor Burden of Solid Tumors In Vivo.
To examine if the ability of IODVA1 to reduce oncogene-driven cell proliferation can be translated in vivo, applicants tested its efficacy on one breast cancer and one lung cancer xenograft mouse model. The breast cancer model utilizes the human triple negative breast cancer MDA-MB-231 cells. Cells were orthotopically injected into the right and left inguinal mammary glands of immunodeficient female mice. Tumor-bearing mice then received an intraperitoneal (IP) injection of 250 μL of 1 min IODVA1 every other day, for an average dose of 3.5 mg/kg. Dosage regimen was not optimized, and it is expected that better results are obtained if IODVA1 dosage is increased. Four weeks post treatment a significant decrease of ≥50% of tumor volume compared to vehicle-treated control mice was observed (
At the end of the four-week treatment when mice were euthanized, peripheral blood was collected via cardiac puncture to check the effect of IODVA1 on white and red blood cells of treated mice for any sign of toxicity. There was no significant difference in WBC, neutrophils, lymphocytes, monocytes, RBC, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin volume (MCHV), red cell distribution width (RDW), platelets, and mean platelet volume between blood of drug- and vehicle-treated mice (
The lung cancer mouse model was generated by injecting the lung cancer H2122 cells at the right and left flanks of NSG mice. These cells harbor the KRASG12C mutation and form aggressive tumors. Mice were treated with IODVA1 or with vehicle control every other day for 14 days. Here also, a significant decrease in tumor volume was observed for mice treated with IODVA1 (
Vehicle-treated H2122 tumors were dense with tumor cells with a high mitotic rate (arrows in
Taken together, applicant's in vivo data suggest that IODVA1 is efficacious at treating solid tumors, including Ras-driven solid tumors, likely by increasing tumor cell apoptosis and decreasing cell proliferation. In addition, IODVA1 administration does not result in adverse effects on bone marrow function due to lack of biologically relevant peripheral blood count changes.
Ras is at the center of many protein-protein interactions that are key to proper cell signaling and normal physiology. Single point mutations or loss of an otherwise fine-tuned regulation lead to a decrease in its GTPase activity and are frequently found in cancer and in a class of diseases called RASopathies (Rauen, 2013; Stephen et al., 2014; Tajan et al., 2018; Tidyman and Rauen, 2009a). Despite decades long efforts in academia and private pharmaceutical companies, targeting Ras has been extremely challenging to translate to therapeutics. Excellent reviews discussing this topic and the various approaches taken to target Ras have been reported in the literature (Marin-Ramos et al., 2018; Ostrem and Shokat, 2016; Spencer-Smith and O'Bryan, 2017; Spiegel et al., 2014). High-resolution structures did not reveal a deep druggable cavity and there are no known Ras agonists or antagonists to provide a starting scaffold to build a chemical discovery process. Despite these difficulties, several low molecular weight chemicals have been reported to bind to Ras albeit with low affinity (Cox et al., 2014; Maurer et al., 2012; Quevedo et al., 2018; Shima et al., 2013; Spiegel et al., 2014; Sun et al., 2012). Inhibiting Ras-binding to its downstream effectors also has been challenging since Ras makes flat and large interfaces (˜1,000-2,000 Å2) with Raf kinase, PI-3 kinase, and RalGDS formed by the juxtaposition of β-strands from each partner protein. Because GTP-loading is such an essential step in Ras signaling, recent efforts are aiming at interfering with the GDP-GTP exchange by targeting the Ras/Sos interface (Evelyn et al., 2015; Evelyn et al., 2014; Maurer et al., 2012; Schopel et al., 2013; Spiegel et al., 2014; Sun et al., 2012). In general, it remains to be seen if these small molecules are efficient in in vivo models of oncogenic Ras, if their true mechanism of action is by directly inhibiting Ras, or if they represent a molecular scaffold on which more potent molecules can be derived. If the task seems insurmountable, recent reports suggest that it is not impossible. For example, the finding that compound 3144 binds to all Ras isoforms and displays anti-tumor activity in xenograft mouse cancer models is encouraging (Welsch et al., 2017). And so is the recent advance toward covalent in vivo inhibition of KRASG12C, a common mutation in lung adenocarcinoma (Janes et al., 2018).
In attempting to exploit a cavity identified in the crystal structure of the GTP-bound form of RasG60A (Ford et al., 2005; Ford et al., 2009) and to target it by a small molecule that restraints the switch 1 loop from coordinating the Mg2+-ion and adopting a signaling conformation, applicants were able to combine in silico screening with cell proliferation and colony formation assays, and identify NSC124205, an inhibitor of the proliferation of Ras-transformed cells at low micromolar concentration. However, HPLC/MS analysis showed that NSC124205 is a mixture of at least three components. Other investigators have reported a high percentage of commercially available chemicals that failed quality control by UPLC (Corsello et al., 2017). This prompted Applicants to identify the active ingredient in the NSC124205 mixture responsible for the antiproliferative activity. Following a simple synthesis scheme in an attempt to resynthesize NSC124205, applicants synthesized a novel small molecule with drug-like properties we termed IODVA1. HPLC and MS analysis indicate that IODVA1 corresponds to peak 1b of the NSC mixture. Given that peak 1c is a tautomer of 1b and given that peak 1a is inactive in cellular assays it is believed that IODVA1 is the active ingredient in NSC124205.
The high sensitivity of RASWT expressing MCF7 and T47D cells and oncogenic Ras expressing MDA-MB-231 to IODVA1 suggest that IODVA1 targets a node used by Ras to regulate cell survival and cell spreading and movement including lamellipodia formation. One such node is the small GTPase Rac. The biochemical observations that IODVA1 decreases Rac activation and signaling, e.g. PAK1 activity (
In conclusion, applicants identified a di-pyridine guanidinobenzimidazole derivative with in vitro and in vivo activity against cell lines xenograft models of cancer. IODVA1 significantly reduces the proliferation of a variety of cancer model cells with diverse genetic lesions and it does so at a low micromolar concentration. The in vivo data show that it inhibits tumor growth by increasing apoptosis and/or decreasing proliferation of cancer cells. The cellular studies and studies of peripheral blood from mice treated with IODVA1 for four weeks (12 doses) revealed no apparent toxicity and suggest that IODVA1 may be specific to transformed cells. Applicant's cellular and in vivo data are consistent with IODVA1 targeting Rac signaling.
EXPERIMENTAL PROCEDURESPlasmids, Cell Lines, and Reagents. MDA-MB-231, MCF7, T47D, MCF10A and HEK239T cells were obtained from ATCC and have since been verified by DNA Diagnostics Center (Fairfield, Ohio) during the course of these studies. NIH-3T3 fibroblasts were a kind gift from Dr. Susanne I. Wells, ST8814 cells were a kind gift from Dr. Nancy Ratner, A549 and H292 cells were a kind gift from Dr. Jeffrey Whitsett. MDA-MB-231 cells were maintained in Improved MEM media (Invitrogen) supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% amphotericin B. MCF10A were maintained in DMEM/F12 (Invitrogen) supplemented with 5% horse serum, 20 ng/mL EGF, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin and 10 μg/mL insulin. MCF7 and T47D were maintained in DMEM, supplemented with 10% FBS and 10 μg/mL insulin. HEK293T and NIH/3T3 cells were maintained with DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. ST8814, A549, and H292 cells were grown in RPMI supplemented with 10% FBS. Cells were treated with the indicated concentrations of compounds. Control cells were treated with equal volumes of diluent only. The following antibodies were used -GAPDH (GeneTex GTX627408, 1:5000), pERK1/2 T202/Y204 (CST 4370, 1:2000), total ERK1/2 (CST 4696, 1:2000), pAKT 5473 (CST 9271, 1:1000), pPAK1 T423/pPAK2 T402 (CST 2601S, 1:1000), PAK1/2/3 (CST 2604, 1:2000), PAK1 (CST 2602, 1:1000), pPAK4 S474/pPAK5 S602/pPAK6 5560 (CST 3241, 1:1000), PAK4 (CST 62690, 1:1000), anti-mouse Eu (Molecular Devices R8205), anti-rabbit Eu (Molecular Devices R8204), anti-mouse-HRP (CST 7076, 1:5000), and anti-rabbit-HRP (CST 7074, 1:5000), Ki67 (Abcam, IHC 1:100), cleaved caspase 3 (Asp175, CST 9661, IHC 1:100), goat anti-rabbit Alexa Fluor 568 (Abcam, IF 1:500). Fluorescent phalloidins and DAPI were from Invitrogen.
Computational Virtual Screening.
Virtual Screening was performed to identify candidate molecules that would stabilize the open conformation of Ras by targeting the cleft situated between the switch 1 and the triphosphate nucleotide in the crystal structure of HRasG60A (PDB ID: 1XCM) (
A subset of 118,500 drug-like synthetic compounds from the NCI/DTP Open Chemical Repository (http://dtp.cancer.gov) was used for virtual screening. These compounds were derived from the NCI Plated 2007 deposited in the Zinc library (http://zinc.docking.org/catalogs/ncip) by using chemoinformatic filters as described in (Irwin and Shoichet, 2005; Irwin et al., 2012; OpenEye Scientific Software). 3D-structures for the resulting subset of 118,500 compounds were downloaded from ZINC. Gesteiger partial charges were used for both the Ras and chemical compounds. Screening was performed in three stages, using increasingly stringent parameters and gradually more extensive sampling. The latter was achieved by increasing the number of energy evaluations (from 2×105 to 1×107), Genetic Algorithm runs (from 20 to 50) and population size (from 75 to 150), as previously discussed (Biesiada et al., 2011) After initial fast screening, 30,000 top candidates with the highest estimated binding affinities were retained, and subsequently re-scored using improved sampling in the refinement stage. 3,000 top hits were then re-scored using extensive sampling and assessed further to select candidates for experimental validation.
These candidate compounds were ranked based on their estimated binding affinities and entropy of clustering of docking poses in multiple runs of docking simulations resulting in a set of 299 NCI library hits that had entropy of docking poses below 0.2 and predicted median binding constants of less than 10 μM. These top hits were subsequently clustered based on their chemical similarity using Chemmine (http://chemmine.ucr.edu) to further select candidates for experimental validation, while avoiding over-representation of some classes of chemicals, and to visually analyze candidate compounds.
From this joint set, a subset of 40 compounds representing different clusters of chemicals were selected for experimental screening based upon assessment of drug-like properties, similarity to classes of compounds often identified in virtual screening as false positives, and availability of compounds from the NCI/DTP Open Chemical Repository (http://dtp.cancer.gov).
MTS assays. The colorimetric CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS, Promega) was used to determine the number of viable cells and evaluate effect of compounds on cell proliferation. Measurements were made as per supplier's protocol. Assays were performed by adding 10 μL of MTS reagent directly to the culture wells followed by 1 h incubation at 37° C. The amount of formazan obtained at the end of incubation was measured by absorbance at 490 nm in a 96-well plate reader (Molecular Devices; Sunnyvale, Calif.). Each 96-well plate had a set of four wells containing medium only and a set of four wells containing cells treated with DMSO vehicle control. Background absorbance was first evaluated from the set of wells containing medium only, averaged, and subtracted from each well. Background corrected absorbance readings were then normalized to and expressed as a relative percentage of the plate averaged DMSO vehicle control. Each experiment was repeated twice per cell line and order of compound arrangement in plates was randomized in different experiments.
Chemical Synthesis.
All chemicals, reagents and solvents were purchased from Sigma-Aldrich, Ark Pharm Inc., and Fisher Scientific. Indicated reaction temperatures refer to those of the reaction bath, while room temperature (RT) is noted as 25° C. Analytical thin layer chromatography (TLC) was performed with glass backed silica plates (20×20 cm, pH=5, MF254). Visualization was accomplished using a 254 nm UV lamp. 1H- and 13C-NMR spectra were recorded on a Bruker Avance 400 MHz spectrometer using solutions of samples in methanol-d6. Chemical shifts are reported in ppm with tetramethylsilane as standard. Data are reported as follows: chemical shift, number of protons, multiplicity (s=singlet, d=doublet, dd=doublet of doublet, t=triplet, q=quartet, b=broad, m=multiplet). All compounds were characterized by 1H-NMR, 13C-NMR and high resolution mass spectroscopy (HRMS).
2-guanidinobenzimidazole (500 mg, 2.86 mmol) and α-pyridoin (1.22 g, 5.72 mmol) were dissolved in N—N-dimethylformamide (5 mL). After the addition of glacial acetic acid (0.2 ml, 3.4 mmol), the reaction was stirred at 85° C. for 48 h. The reaction was cooled then quenched with water, the pH was neutralized, and the aqueous solution was extracted three times with ethyl acetate. The organic layers were combined and washed with water and brine then dried over Na2SO4 and filtered using filter paper. Silica (2 g) was added before concentrating the solution under reduced pressure. The solid was loaded onto a column then purified with flash chromatography using 1:10 methanol: methylene chloride gradient. The targeted fractions (Rf=0.5) were collected and reduced under pressure to yield 111 mg (0.30 mmol, 11%) of the desired product as a brown-gold solid.
1H NMR (400 MHz, MeOD-d6): δ=7.07 (m, 3H), 7.33 (m, 2H), 7.51 (m, 2H), 7.63 (m, 1H), 7.85 (m, 1H), 8.05 (m, 1H), 8.19 (m, 1H), 8.33 (m, 1H), 8.53 (d, 1H).
13C NMR (400 MHz, MeOD-d6): δ=113.89, 122.04, 122.19, 123.16, 124.17, 124.74, 128.85, 129.53, 137.93, 138.93, 148.65, 149.81, 150.36, 150.60, 152.89, 156.43, 157.79, 161.23.
HRMS-ESI: [M+H]+ (C20H16N7O ): calculated: m/z=370.1411. Found: m/z=370.1409
Synthesis of NIRA2 2-((1H-benzo[d]imidazol-2-yl)imino)-5,5-di(pyridin-2-yl)imidazolidin-4-one and 2,2,5-tri(pyridine-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one (NIRA2)1,2-di(pyridin-2-yl)ethane-1,2-dione (5 g, 23.6 mmol) and 2-(1H-benzo[d]imidazol-2-yl)guanidine (5.37 g, 30.6 mmol) in DMSO (80 mL) was heated to 110° C. 1 M KOH solution in water (28.3 mL) was added dropwise and the mixture was kept at 110° C. for 30 min. The mixture was poured into water (300 mL) and acidified with 1 N HCl solution to pH=6. The mixture was extracted with EtOAc (3×100 mL). The combined organic phase was dried with anhydrous sodium sulfate, filtered and concentrated in vacuum. The crude product was triturated with EtOH/DMF (5:1, 50 mL) and filtered to give 1 g of 2-((1H-benzo [d]imidazol-2-yl)imino)-5,5-di(pyridin-2-yl)imidazolidin-4-one as a light yellow solid and 0.72 g of 2,2,5-tri(pyridin-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5] triazin-3(5H)-one, which was further purified by reverse phase HPLC (column: Kromasil C18 (250*50 mm*10 um); mobile phase: [water (10 min NH4HCO3)-ACN]; B %: 15%-40%, 10 min) to afford 2,2,5-tri(pyridin-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one (NIRA2) (100 mg, 1% yield). LCMS: m/z found 459.1 [M+H]+
2,2,5-tri(pyridin-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one (NIRA2_P1) and 2,2,5-tri(pyridin-2-yl)-1,2-dihydrobenzo[4,5]imidazo [1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one (NIRA2_P2)100 mg of the mixture of enantiomers was separated by SFC (column: DAICEL CHIRALCEL OJ (250 mm*30 mm, 10 μm); mobile phase: [0.1% NH3H2O IPA]; B %:43%-43%, min) to give 22,25,25-tris(2-pyridyl)-29,30,31,32,33-pentazatetracyclohexadeca-(8),1(9), 16,21(29),24(31)-pentaen-23-one (NIRA2_P1) (faster eluting enantiomer, 35.75 mg, 36% yield) as white solid and 22,25,25-tris(2-pyridyl)-29,30,31,32,33-pentazatetracyclohex adeca-(8),1(9),16,21(29),24(31)-pentaen-23-one (NIRA2_P2) (slower eluting enantiomer, 37.01 mg, 37% yield) as white solid.
2,2,5-tri(pyridin-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one (NIRA2_P1)MS: m/z found 459.2 [M+H]+; HPLC: Rt: 1.751 min; SFC: Rt=1.734 on OJ column; 1H NMR (400 MHz, MeOD): δ 8.62 (d, J=4.8 Hz, 1H), 8.50 (d, J=4.8 Hz, 1H), 8.42 (d, J=4.8 Hz, 1H), 7.86-7.84 (m, 4H), 7.51-7.41 (m, 5H), 7.36-7.28 (m, 2H), 7.20 (d, J=8.0 Hz, 1H), 7.10 (t, J=7.6 Hz, 1H), 7.03 (t, J=7.6 Hz, 1H)
2,2,5-tri(pyridin-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one (NIRA2_P2)MS: m/z found 459.2 [M+H]+; HPLC: Rt: 1.757 min; SFC: Rt=1.893 min on OJ column; 1H NMR (400 MHz, MeOD): δ 8.62 (d, J=4.8 Hz, 1H), 8.50 (d, J=4.8 Hz, 1H), 8.42 (d, J=4.8 Hz, 1H), 7.87-7.84 (m, 4H), 7.52-7.41 (m, 5H), 7.36-7.28 (m, 2H), 7.20 (d, J=8.0 Hz, 1H), 7.10 (t, J=7.6 Hz, 1H), 7.03 (t, J=7.6 Hz, 1H).
Alternate Synthetic Route for NIRA2:
To a mixture of 2-(1H-benzo[d]imidazol-2-yl)guanidine (2 g, 11.4 mmol) and pyridine-2-carbaldehyde (1.83 g, 17.1 mmol) in EtOH (40 mL) was added piperidine (0.8 mL, 7.99 mmol) under N2. The mixture was stirred at 100° C. for 16 hours. The resulting white solid was collected by filtration and washed with EtOH (30 mL), dried in vacuo to give 4-(pyridin-2-yl)-3,4-dihydrobenzo[4,5]imidazo[1,2-a][1,3,5] triazin-2-amine (2.7 g, 89% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 8.56 (d, J=4.4 Hz, 1H), 8.09 (s, 1H), 7.82 (t, J=7.0 Hz, 1H), 7.38-7.35 (m, 1H), 7.25-7.22 (m, 2H), 6.95-6.88 (m, 2H), 6.83-6.77 (m, 2H), 6.44 (brs, 2H).
2,2,5-tri(pyridine-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one (NIRA2)To a mixture of 4-(pyridin-2-yl)-3,4-dihydrobenzo[4,5]imidazo[1,2-a][1,3,5]triazin-2-amine (200 mg, 0.76 mmol) and 1,2-bis(2-pyridyl)ethane-1,2-dione (210 mg, 0.99 mmol) in EtOH (2 mL) and DMSO (4 mL) piperidine (38.81 mg, 0.46 mmol) was added under N2. The mixture was stirred at 120° C. for 16 hours. The mixture was quenched with water (40 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude was purified by trituration with EtOAc (30 mL), filtered, yellow solid was dried in vacuo to give 2,2,5-tri(pyridine-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one (80 mg, 23% yield) as yellow solid. 1H NMR (400 MHz, DMSO_d6): δ10.43 (brs, 1H), 8.59 (d, J=4.0 Hz, 1H), 8.49 (d, J=4.0 Hz, 1H), 8.38 (d, J=4.0 Hz, 1H), 7.88-7.83 (m, 4H), 7.66 (s, 1H), 7.42-7.31 (m, 7H), 7.00-6.95 (m, 2H).
Immunoblotting.
Cells were lysed in RIPA buffer, supplemented with 1% SDS, protease (Sigma P8340) and phosphatase inhibitors (Roche 04906845001). Lysates were separated on 12%, 15% or 4-20% SDS-PAGE and transferred onto PVDF membrane (Bio-Rad TurboBlot). Membranes were blocked in 5% BSA in TBS-T (0.05%) and incubated overnight with primary antibodies. Blots were washed, probed with the appropriate secondary antibodies and processed with ECL (film or Bio-Rad chemiluminescent system) or imaged on the SpectraMax i3 platform with ScanLater module (Molecular Devices).
Retrovirus Production and Transduction.
pBabe puro HRasG12V plasmid was from Addgene (#9051). HRasWT was made by reverting G12V to G using site-directed mutagenesis. Plasmids were verified by Sanger Sequencing (CCHMC DNA Core). Retroviral supernatants were produced by transfecting HEK293T cells with pBabe-puro plasmids with pCL-Eco in 1:1 ratio using Calcium Phosphate method (Trono Lab). Supernatants were harvested 24 and 48 h post-transfection and filtered. NIH/3T3 cells were transduced overnight in the presence of polybrene (10 μg/mL) and selected with puromycin (3 μg/mL). Retroviral production and manipulation were performed in BSL-2 facilities.
Anchorage-Independent Growth Assays.
Bottom agar layer was prepared by mixing 2× complete DMEM with 1% Noble agar (BD) in a 12-well plate for a final concentration of 0.5% and allowed to solidify. For the top agarose layer for each well, 2,500 cells in 2× compete DMEM were resuspended in 0.6% low-melting agarose (IBI Scientific) and layered over the bottom agar layer. The next day, 100 μL of complete growth media containing test compounds was overlaid the upper layer to prevent desiccation. Media was refreshed twice weekly. After 21 days, colonies were stained with 0.1% p-iodonitro tetrazolium violet (Sigma-Aldrich), imaged using an EVOS microscope (Life Technologies), and counted.
Spheroid Formation Assay.
Spheroids were formed by the hanging drop method or using ultra-low attachment (ULA) plates (Corning). For the hanging drop assay, cells were trypsinized, resuspended at 25,000 cells/mL in complete media containing vehicle (DMSO), or the indicated concentrations of IODVA1. and plated in 25 μL drops on an inverted lid of a 10-cm dish. The dish was filled with 7 mL PBS, the lid replaced and incubated for 3-5 days. For mechanical testing of spheroid compaction, spheroids were first imaged on EVOS microscope, repeatedly pipetted 7-9 times, then imaged again to assess spheroid disruption. For the ULA-based spheroid formation, 5,000 cells were resuspended in 500 μL of complete media containing vehicle (DMSO) or the indicated concentrations of IODVA1 and plated in 24-well ULA plates (Corning). Spheroid formation was monitored daily and imaged using EVOS microscope (Life Technologies). For assessment of attachment-free proliferation, the contents of the well were transferred into an Eppendorf tube, centrifuged at 100×g for 5 min, dissociated into a single cell suspension using trituration and Accutase (Invitrogen) treatment at room temperature and counted using trypan blue exclusion.
Active GTPase Binding Assays:
Levels of active Ras, Rac, Cdc42 and RhoA were determined using the Active Ras Pull-Down and Detection Kit, Active Rac Pull-Down and Detection Kit (Thermo Scientific) and RhoA/Rac1/Cdc4 Pull-down Activation Assay Combo Biochem Kit (Cytoskeleton). Cells were cultured and treated as indicated and lysed in buffer provided by the manufacturer. Clarified whole cell lysates were incubated with recombinant GST-Raf1-RBD (for active Ras) (amino acids 1-149) and glutathione beads (both supplied by the manufacturer), GST-Rhotekin-RBD (for active RhoA), GST-PAK-GBD (for active Rac and Cdc42) for 1 h at 4° C., washed and the resulting complexes eluted from the resin by boiling in 2×SDS sample buffer. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose and the level of active GTPase relative to input lysate were determined by immunoblotting analysis using the anti-Ras, anti-Rac, anti-Cdc42, or anti-RhoA antibody supplied by the manufacturer.
Kinase Assay:
The Reaction Biology (http://www.reactionbiology.com) HotSPot miniaturized radioisotope filter binding assay platform was used to measure the activity of 369 wild-type kinases in presence of a single IODVA1 dose (Anastassiadis et al., 2011). In brief, for each reaction, kinase and substrate were mixed in a buffer containing 20 min HEPES (pH 7.5), 10 min MgCl2, 1 min EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 min Na3VO4, 2 min DTT, and 1% DMSO. IODVA1 was then added to a final concentration of 0.5 μM to each reaction mixture. After 20 min incubation at room temperature, reaction was initiated by adding ATP (Sigma-Aldrich) and [γ-32P]-ATP (PerkinElmer, specific activity of 10 Ci/L). Reactions were incubated for 2 h at room temperature and spotted onto P81 ion exchange cellulose chromatography paper (Whatman). Filter paper was washed in 0.75% phosphoric acid to remove excess ATP. The percent remaining kinase activity relative to a vehicle-containing (DMSO) kinase reaction was calculated for each kinase/IODVA1 pair. Each kinase inhibition assay was done in duplicates and averaged. Data were processed and analyzed in Excel.
Immunofluorescent Analysis of Actin Cytoskeleton:
MDA-MB-231 cells were seeded at a density of 2×104 cells per chamber in an 8-chamber glass slide with or without EGF (5 ng/ml) for 10 min After treatment with 1 uM IODVA1, cells were fixed in 4% paraformaldehyde, permeabilized in 0.2% TritonX-100, then stained with phalloidin conjugated to Alexa Fluor568 (1:100) and ProLong Gold with DAPI (both Life Technologies, Thermo-Fisher). Staining was visualized with a Nikon MR confocal microscope.
Immunofluorescence and Microscopy:
For assessment of lamellipodia initiation and maturation, assays were performed in two ways. 1) MDA-MB-231 were plated on fibronectin-coated coverslips in serum-free media for 4 h, followed by 1 h incubation with IODVA1 (0-3 μM) in serum-free media. Cells were then EGF stimulated (50 ng/mL) for 30 min to induce lamellipodia formation. Cells were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton-X 100 and stained with Phalloidin Alexa Fluor 568 or 594 and mounted in ProLong Gold DAPI (Life Technologies). 2) MDA-MB-231 cells were seeded at a density of 2×104 cells per chamber in an 8-chamber glass slide with or without EGF (5 ng/ml) for 10 min. After treatment with 1 μM IODVA1. cells were fixed and processed as in 1). Staining was visualized with a Nikon A1R confocal microscope. 10-random fields were imaged and analyzed for lamellipodia formation. For assessment of circular dorsal ruffle formation, NIH-3T3 cells were processed as in (Steffen et al., 2013). Briefly, cells (4×104) were plated on fibronectin-coated coverslips, serum-starved for 4 hours and treated with IODVA1 (0-3 μM) in serum-free media for 1 hour. Ruffling was induced with PDGF BB (50 ng/mL, Peprotech) for 10 min. Cells were fixed in 4% paraformaldehyde and processed for immunofluorescence microscopy as above. Around 150 cells were counted per treatment group per experiment. For assessment of stress fibers, cells were plated on fibronectin-coated coverslips for 4 hours in serum-free media, incubated with IODVA1 (0-3 μM) for 1 h, fixed and processed with fluorescent phalloidin as before.
For cell spreading, MDA-MB-231, MCF7 and T47D were seeded on fibronectin-coated coverslips in serum-free media. After 10 min, IODVA1 (0-3 μM) was added in serum-free media. After 30 min (40 min total), cells were fixed in 4% paraformaldehyde and imaged using bright field microscopy (EVOS, Life Technologies). Six random fields were imaged under 20× objective and cell area of individual cells were quantified to assess the degree of spreading (ImageJ). Over 300 cells were counted per treatment group per experiment.
In Vivo Analysis Using MDA-MB-231 Xenograft Mouse Model:
For xenograft studies, 1×106 MDA-MB-231 cells were suspended in PBS and injected into each inguinal mammary fat pad of nulliparous, 10-week old female athymic nude mice. Tumors were measured weekly with digital calipers and volume was calculated as [(π/6))×L×W2] (Euhus et al., 1986; Tomayko and Reynolds, 1989). Treatment began eight weeks post-injection, when tumors reached 200 mm3. Mice received intraperitoneal injections of 250 uL of diluent (5% DMSO in PBS) or 1 min of compound IODVA1 three times weekly for 4 weeks, for an average drug dose of 3.5 mg/kg. At necropsy, the mice were weighed and the tumors were excised, measured, weighed, fixed in 4% paraformaldehyde, and embedded in paraffin. Peripheral blood was collected by cardiac puncture and analyzed with a Hemavet (Drew Scientific, Miami Lakes, Fla., USA) for complete blood counts. Usage and handling of mice were performed with the approval of the Cincinnati Children's Institutional Animal Care and Use Committee. All mice were housed in specific pathogen free housing with ad libitum access to food and water.
Histology:
Tissues were fixed in 4% paraformaldehyde then paraffin embedded tissues were cut into 5 μm sections. Tissues were stained with either hematoxylin and eosin (H&E) or by immunofluorescence. Tissue sections were subjected to sodium citrate antigen retrieval, blocked with 10% normal goat serum, and incubated with antibodies to Ki67 (1:100, Abcam) or cleaved caspase 3 (Asp175, 1:100, Cell Signaling) then goat anti-rabbit:Alexa568 (1:500, Abcam). Tissues were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and coverslipped with VectaShield HardSet (Vector Labs) Images were acquired by confocal microscopy (Nikon) and immunofluorescence analysis was performed using Image J.
In Vivo Analysis of H2122 Xenograft Mouse Model.
Human lung cancer cell line NCI-H2122 (ATCC) harboring biallelic KRASG12C mutations were cultured in RPMI-1640 supplemented with 10% FBS. For injections, NCI-H2122 cells were trypsinized and suspended at a concentration of 2×107 cells/mL in 50% Matrigel (Corning). 100 μl of cell suspension was injected subcutaneously into 6-week old NOD.Cg-Prkdcscid Il2rgtmIWjI/SzJ (NSG) mice. Beginning 10 days post-xenograft, mice received intraperitoneal injections every other day for 14 days (7 injections total). A 200 mM IODVA1 stock solution in DMSO was freshly diluted 1:19 in DMSO then subsequently diluted 1:9 in PBS to achieve a 1 min final concentration in 10% DMSO. Mice were injected with 250 μl of vehicle (10% DMSO) or IODVA1 (1 mM). Tumor size was measured using digital calipers and tumor volume estimated using the formula (length×width2)/2. For immunostaining, paraffin-embedded sections were stained with Ki-67 antibody (clone SP6, ThermoFisher) at a dilution of 1:1,000. DAB stained slides were counterstained with nuclear-fast red stain. For quantification of Ki-67 positive cells, random fields were imaged at 200× magnification and quantified using Image J software. A minimum of 20 random fields were used for quantification. Scale bar=100 μm.
Notes: Vehicle treated mice had readily visible mitotic cells. IODVA1 had increased intratumoral fibrosis. Both vehicle and IODVA1 tumors had centralized necrosis, typical for subcutaneous tumors at that size.
Inhibition of Rac Activity
Rac GTPases (Rac1, Rac2, Rac3, and RhoG) are tightly regulated signaling switches that mediate inputs from various receptors and oncogenes to regulate growth, apoptosis, cell-cell and cell-matrix interactions in response to growth factors such as EGF, PDGF, and HGF. Regulation of the actin cytoskeleton, which plays a key role in cell shape, polarity, division, migration and metastasis, is a major function of Rac, as it promotes membrane ruffling and formation of lamellipodia and circular dorsal ruffles (Etienne-Manneville, 2002; Jaffe & Hall, 2005; Bustelo, 2018; Ridley & Hall, 2015; Ridley, 1992; Steffen, 2013). Rac also controls cell cycle progression and cell survival, integrin-mediated adhesion, and is required for Ras transformation (Coleman, 2004; Kiosses, 2001; Sundaresan, 1996; Mack, 2011; Qiu, 1995). Furthermore, Rac is pivotal in most aggressive types of leukemias (Thomas, 2007; Somervaille, 2006; Wei, 2008; Sengupta, 2010; Skorski, 1998; Mizukawa, 2011; Bassermann, 2002; Nieborowska-Skorska, 2012). Thus, Rac has been associated with pro-tumorigenic functions and linked to the development of cancer. Moreover, Rac is characteristic of resistance to chemo-, radio-, and targeted-therapies and associated with persistence of leukemic stem cell (Jaffe & Hall, 2005; Loirand, 2010; Mulloy, 2010; Newey, 2005; Sahai, 2002; Vigil, 2010; Porter, 2016; Zandvakili, 2017; Cardama, 2018).
Reducing Rac activity, specifically in cancer cells, is desirable and is an active area of research. However, no small molecule inhibitor of Rac signaling is in clinical use despite the many efforts. Rac activity is regulated by an intricate and well-orchestrated set of proteins comprised of guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine dissociation inhibitors (GDIs). RacGEFs activate Rac by exchanging the bound GDP to GTP to initiate signaling while GAPs deactivate Rac by increasing the rate of GTP hydrolysis to arrest signaling. GDIs extract Rac from membranes, thereby preventing it from signaling. When activated, Rac binds to and activates downstream effectors such as the p21-activated kinases (PAK1/2/3), which in turn activate pro-survival pathways and actin-regulating proteins. Rac regulators and effectors are themselves subject to tight regulation. For example, Vav proteins (Vav1, Vav2, and Vav3) are multi-domain tyrosine phosphorylation-dependent RacGEFs. Phosphorylation of specific tyrosine residues releases an N- and C-terminal autoinhibition mechanism allowing Rac to access the Dbl-homology (DH) domain necessary for the GTP-exchange reaction. Therefore, finding a small molecule inhibitor of Rac itself or its activator, such as the Vav proteins, will provide an effective strategy for treatment of malignancies with aberrant Rac signaling.
In this study, applicants reveal the mechanism of action (MoA) of IODVA1, a 2-guanidinobenzimidazole derivative identified as the active ingredient in NSC124205. Initial characterization of IODVA1 showed that it is not a kinase inhibitor but that it prevents lamellipodia and circular dorsal ruffle formation at low concentrations and within minutes of cell exposure. It also decreases cell-cell and cell-extra cellular matrix interactions and reduces growth of Ras-driven tumors. These properties and the specificity of IODVA1 to oncogene expressing cells hinted that it targets Rac activation. The Applicants used in vitro and in vivo leukemic models of the chimeric BCR-ABL1 oncoprotein B-cell acute lymphoblastic leukemia (Ph+B-ALL) to study the MoA of IODVA1. BCR-ABL1 B-ALL models are well-suited for this endeavor for several reasons. First, BCR-ABL B-ALL is a single-driver genetic model. Expression of BCR-ABL, which has constitutive kinase activity is sufficient to promote the growth advantage of leukemic cells. When expressed, BCR-ABL1 activates a variety of pathways including the Ras-mitogen-activated protein kinase (MAPK) leading to abnormal cell proliferation, the Janus-activated kinase (JAK)-STAT pathway leading to impaired transcriptional activity, and the phosphoinositide 3-kinase (PI-3K)/AKT pathway resulting in prolonged survival (Cilloni, 2012). In addition, expression of p190- or p210-BCR-ABL activates Rac signaling pathways to regulate leukemogenesis (Skorski, 1998; Thomas, 2008; Harnois, 2003; Sahay, 2008) and deleting Rac2 or the combination of Rac1 and Rac2, impairs myeloid leukemogenesis induced by p210-BCR-ABL expression in the hematopoietic stem and progenitor cell compartment (Thomas, 2007; Sengupta, 2010). Second, seemingly complex pathways activated by BCR-ABL all depend on the deregulated kinase activity of BCR-ABL (Lugo, 1990) and ABL1-tyrosine kinase inhibitors (ABL1-TKIs), e.g. imatinib, are used as first-line therapy. Thus, imatinib can be used as positive control to assess the efficacy of IODVA1. Third, despite their tremendous success in treating B-ALL in the clinic, appearance of mechanisms of TKI-dependent and -independent resistance limited their efficacy (Arrigoni, 2018; Hamilton, 2012). Thus, there is an unmet need for novel treatments of patients with TKI-resistant leukemia and treatments that prevent leukemic-cell persistence.
Here, the Applicants show that IODVA1 binds tightly and inhibits Vav3, consequently leading to the deactivation of Rac and of its downstream signaling and to the induction of apoptosis specifically in BCR-ABL expressing cells in vivo and in vitro. Further, the Applicants show that IODVA1 prolongates the survival of a mouse model of TKI-resistance and reduces its leukemic burden long after treatment was withdrawn. The Applicants also show that IODVA1 is effective in decreasing proliferation and survival of relapsed and de novo primary patient-derived cells. It is believed that IODVA1 is the first inhibitor of a RhoGEF with in vivo activity against xenograft mouse models of cancer. The Applicants findings have direct implications for overcoming TKI-resistance in the clinic and for treating cancers where Vav3 is a target, including Ras-driven cancers.
Results
IODVA1 specifically targets BCR-ABL B-ALL cells in vitro. To confirm that IODVA1 is specific for oncogene-expressing cells, we tested its efficacy on the proliferation and survival of CD34+ human peripheral blood mononuclear cells transduced with retroviral bicistronic p190-BCR-ABL or Mieg3 empty vector (Williams, 2000). As expected, expression of BCR-ABL increased cell proliferation (
IODVA1 prevents leukemia-related death and significantly decreases the leukemia burden in a BCR-ABL-induced leukemic murine model. To test if IODVA1's potency on cells can be recapitulated in vivo, applicants probed its efficacy on a murine model of p190-BCR-ABL induced B-ALL and compared it to imatinib, an ABL1-TKI with well-characterized MoA in mouse models of Ph+B-ALL used as first-line therapy in Ph+-induced malignancies in the clinic. C57Bl/6 mice were used as donors and/or recipients of transduction/transplantation model. Mouse low-density bone marrow (LDBM) cells were transduced with a MSCV-driven bicistronic retroviral vector (MSCV-IRES-EGFP) encoding p190-BCR-ABL. Transduced LDBM cells (1×106) were intravenously transplanted into lethally irradiated C57Bl/6 mice. Mice were bled post 23 days and GFP+ cells were analyzed by flow cytometry. All mice had developed leukemia by day 28. Leukemic mice were stratified into 5 groups (7 mice per group) and administered either PBS control vehicle, 0.25 or 0.5 min IODVA1. 0.5 min imatinib, or the combination 0.25 min IODVA1+0.5 mM imatinib Vehicle control group had the same DMSO amount (0.1%) as the other groups. Drugs were administered subcutaneously in osmotic pumps for continuous slow release for 4 weeks. Mice survival plot shows that while the control group had died within 7 to 10 days post administration of the PBS vehicle, the low IODVA1 dose (0.25 mM) increased survival by an average of 10 days. Mice treated with imatinib or 0.5 min IODVA1 or the combination were alive for the 4-week duration of the therapy (
IODVA1 Eradicates Leukemic Propagating Activity Assessed by Serial Transplantation.
Despite its significant clinical success, imatinib and, more generally, TKIs do not eliminate leukemic stem/progenitor cells in the bone marrow (BM), which can lead to residual disease, appearance of mechanisms of resistance, and ultimately relapse (Milojkovic, 2009; Bixby, 2009). To determine if IODVA1 eradicates progenitor B-cells with ability to propagate tumors as a functional surrogate of minimal residual disease capable of leukemia relapse, BM cells from vehicle-control and treated mice from
IODVA1 Eradicates TKI-Resistant BCR-ABL B-ALL.
Because IODVA1 has no inhibitory activity against major wild-type kinases including ABL1 and SRC-like kinases, the anti-proliferative activity of IODVA1 towards in vitro and in vivo BCR-ABL B-ALL models and its ability to eradicate residual disease cannot be explained by ABL1 inhibition. To further test this idea, we evaluated the ability of IODVA1 to increase the survival of mice harboring p210-BCR-ABL(T315I). We chose this ABL1 mutant because it is one of the most frequent mutations arising in chronic myelogenous leukemia (CML) patients on imatinib therapy (Gorre, 2001; Azam, 2003; Jabbour, 2006; Nicolini, 2006; Jabbour, 2008). Mice were treated for 4 weeks with two rounds of pumps containing vehicle control, imatinib, or IODVA1. At the end of the 4-week treatment, surviving mice were kept in their cages without any additional treatment. As expected, p210(T315I) mice did not respond to the TKI as all imatinib-treated mice died by day 22, before the end of the treatment (
To assess signaling pathways affected by IODVA1. EGFP+/B220+ LDBM cells were isolated from two-week treated p210(T315I) mice, stained with phospho-antibodies against the pro-proliferative Rac-dependent effectors JNK, PAK, 4EBP, and S6 and the Rac-independent effectors ERK1/2, STAT3, STAT5, p38, and AKT, and analyzed by flow cytometry. IODVA1 resulted in significant decreases in pJNK by 55% (p=0.0029), pPAK by 56% (p=0.0016), p4EBP by 20.3% (p=0.037), and pS6 by 17.8% (p=0.0012), respectively (
IODVA1 Decreases Rac Activity and Downstream Signaling.
Having shown that IODVA1 prevents formation of F-actin suprastructures, such as lamellipodia and circular dorsal ruffles, within minutes of cell incubation (
To test if the decrease in Rac activation is translated into a decrease in its downstream signaling in vitro as was observed with xenograft-derived TKI-resistant cells (
Significantly, IODVA1 decreases Rac downstream pro-survival PAK and decreases inhibitory phosphorylation of pro-apoptotic BAD Ser136 activities within minutes of cell exposure (
Rae-Deficient Cells do not Respond to IODVA1.
To confirm that IODVA1 targets Rac-dependent pathways, we assessed its effects in a Rac2-null background. Rac1Δ/Δ+Rac2−/− murine leukemic cells show severe reduction in Rac1 expression and are deficient in Rac2 (
IODVA1 is an Inhibitor of the RacGEF Vav3.
Rac activity and signaling is regulated by GAPs, GDIs, and RacGEFs. We argued that the decrease in Rac activity might be caused by IODVA1 targeting one Rac regulator. Using biochemical assays, we show that IODVA1 does not stimulate the activity of the Rac negative regulators p50GAP and RhoGDI1 (
IODVA1 Binds to Vav3.
The previous observation suggests that IODVA1 binds to Vav3. To test this hypothesis, we measured its binding affinity (Kd) to recombinant Vav3 and Rac1 using microscale thermophoresis (MST); the catalytic domain (DH/PH) of the RhoGEF LARG served as negative control. We used LARG for two reasons. First, we reasoned that if Rho-activation is not affected by IODVA1 (
Vav3-Deficient Leukemic Cells do not Respond to IODVA1 In Vitro and In Vivo.
To further validate Vav3 as a target of IODVA1, we studied the effects of IODVA1 on leukemic cells from the Vav3-KO (Vav3−/−) mice we published previously (Thomas E K et al., 2007; Thomas E K et al, 2008; Bourgoin S. et al., 1995). We argued that if IODVA1 targets Vav3, then Vav3−/− cells should be insensitive to its action. Wild-type or Vav3−/− murine BM leukemic cells expressing p190-BCR-ABL (EGFP+/B220+) were tested in colony formation assay in the presence of IODVA1 (
Vav3 Rescues IODVA1 Sensitivity.
Next, we reasoned that if IODVA1 targets Vav3, rescuing Vav3−/− leukemic cells by expressing exogenous Vav3 should re-sensitize those cells to IODVA1. We expressed full-length Vav3 or the dominant active ΔCH-mutant (
To test if the results of the previous rescue experiments hold in proliferation assays, we subjected the Vav3 full-length and ΔCH-mutant expressing Vav3−/− leukemic cells to colony formation assays in the presence of IODVA1 (1, 5, and 10 μM) or vehicle control (
Vav3−/− Leukemia do not Respond to IODVA1 In Vivo.
Next, we tested if the lack of response to IODVA1 by Vav3−/− cells holds in vivo. We transplanted wild-type or Vav3−/− LDBM cells transduced with p190-BCR-ABL retrovirus into lethally irradiated C57BL/6 mice, waited for the leukemia to develop, and treated the mice with either vehicle control or IODVA1 administered through osmotic pumps as before. Vav3−/− leukemic mice did not respond to IODVA1 supporting the hypothesis that Vav3 is IODVA1's target in vivo (
IODVA1 Targets Vav3 in MDA-MB-231 Cells and Xenograft Tumors.
Next, we tested if IODVA1 is effective in another model of Vav3-dependent cancer. We chose MDA-MB-231 triple negative breast cancer cells because these cells express high endogenous levels of Vav3 (Chen, 2015) and because Vav3 is an acknowledged target in breast cancer (Chen, 2015; Lee, 2008; Aguilar, 2014; Citterio, 2012; Lorenzo-Martin. 2019). In addition, we have previously shown that these cells are sensitive to IODVA1 and that IODVA1 halts tumor growth and induces apoptosis in MDA-MB-231 xenograft mice (
To test if IODVA1 inhibits Vav3 in vivo, we took advantage of the MDA-MB-231 xenograft tumors we generated (
IODVA1 Decreases Survival of Patient-Derived Leukemia Cells.
Consistent with our findings with peripheral CD34+ BCR-ABL cells (
Cells from relapsed patient #2018-136 with Ph+(BCR-ABL1) were treated with ABL-TKI dasatinib, JAK-inhibitor ruxolitinib, dasatinib and ruxolitinib combination (das+rux), CDK-inhibitor abemaciclib, and IODVA1. Dasatinib (20 nM) reduced the proliferation of #2018-136 cells by 56%; ruxolitinib or abemaciclib (1 μM) had no effect. The das+rux combination resulted in 63% decrease in proliferation, which is likely due to dasatinib's inhibitory action. IODVA1 (0.5 μM) reduced the proliferation of these cells by 78%. When tested in the colony formation assay, IODVA1 (1 μM) reduced the number of colonies by 60% (p=0.001) (
Cells from patient #2017-58 with a dual Ph+(BCR-ABL1) and Ph-like (P2RY8—CRLF2) rearrangement were similarly treated. These cells clearly responded to dasatinib, ruxolitinib, and the combination. IODVA1 was not as potent as it decreased their proliferation by only 40% at 1 μM and had no effect at 0.2 μM (
Our cohort of samples also contained numerous cases of Ph-like disease with a diverse series of genetic aberrations and a few cases of MLL-rearranged B-ALL. These patient cells generally responded positively to IODVA1 (
In conclusion, we have shown that pharmacological inhibition of Vav3 by IODVA1 is an attractive therapeutic strategy to treat Ph+ and TKI-resistant BCR-ABL B-ALL. This strategy should benefit other malignancies where Vav3 is a target, such as other leukemias, poor prognosis breast cancer, skin tumors, prostate cancer, gastric cancer, glioblastoma, or where it is highly expressed. We thus expect IODVA1 to have a broader therapeutic application. In addition, IODVA1 constitutes an exceptional tool to dissect the Vav3/Rac signaling axis. Broadly, RhoGEFs are multi-domain proteins that are regulated by autoinhibition. Consequently, small molecules that stabilize the autoinhibited conformation of RhoGEFs and inhibit their activity could be developed into drugs to treat human cancers.
Plasmids, Cell Lines, and Reagents:
Plasmid set for purification of fixed-arm carrier fusions pMalX (A-E) was a kind gift from Dr. Lars C. Pedersen (NIEHS), pET28b-N9-MBP-mOrange plasmid was from Addgene (#29748), chaperone co-expression plasmid set was from TaKaRa (cat #3340). Primers were from Integrated DNA Technologies (IDT, Inc.). Primer and construct design was performed with NEBuilder Tool. Restriction enzymes, polymerases, cloning assembly kits and competent cells were from New England Biolabs and Invitrogen.
MDA-MB-231 cells were maintained in IMEM (Invitrogen) supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% amphotericin B. B a/F3 cells were cultured in RPMI (GIBCO) supplemented with 10% FBS and IL-3 (10 ng/ml). HEK293T cells were maintained with DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. All cell lines were cultured at 37° C. in a 5% CO2 humidified incubator. Cell viability was assessed by trypan blue exclusion assay as previously described. Cytokines were from Peprotech.
The following antibodies were used: GAPDH (#627408, GeneTex), pERK1/2 (#4370), pAKT (#9271 and #9018), c-Abl (#2862), Cdc42 (#2462), RhoA (#2117), pPAK1/2 (#2601S), pS6 (#4851S), PAK1 (#2602S), pBAD (#4366), and BAD (#9292), anti-mouse HRP (#7076), anti-rabbit HRP (#7074) were from Cell Signaling Technologies, pVav3(Y173) (#ab109544) and total Vav3 (#ab203315) were from Abcam, pJNK (Alexa Fluor 647 conjugated, #562481), p-p38 (PE-conjugated, #612565), Rac2 (#610850), pStat3 (#55385), and pStat5 (Alexa Fluor 647 conjugated, #612599), and B220 APC-Cy7 antibody (#552094) were from BD Bioscience, anti-phosphotyrosine antibody was from Millipore Sigma (#05321), p4EBP1 (PE-conjugated, #12-9107-42) was from Thermofisher Scientific.
Lipids (Phosphatidylserine (PS), Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM), and phosphatidylinositol 4,5-bisphosphate (PIP2) for membrane displacement assays were from Avanti Polar Lipids.
IODVA1 was synthesized from 2-guanidinobenzimidazole and purified as described and Gasilina et al., 2020. Imatinib (#SML1027) was from Millipore Sigma, dasatinib (#S1021), and ruxolitinib (#S1378) from Selleck.
Retroviral and Lentiviral Particle Production, Transduction and Transplantation of Transduced Leukemic Cells:
Production of lentivirus and retrovirus for stable transduction of murine and human cells were done as described previously (Lee et al., 2017). Scrambled and Vav3-specific shRNAs (Sigma-Aldrich MISSION shRNA) were obtained from Cincinnati Children's Lenti-shRNA Library Core. Retroviral and lentiviral vectors, viral transduction of cell lines and mouse LDBM, and transplantation of transduced leukemic cells were previously described (Chang et al., 2012).
For Vav3 rescue experiments, low density bone marrow cells from wild-type (Vav3−/+) or Vav3−/− mice were transduced with bicistronic retroviral vector encoding p190 BCR-ABL1-IRES-YFP (yellow fluorescent protein) and YFP+ cells were sorted 48 h post-transduction. Cells were then transduced with lentiviral particles encoding either empty vector, full-length Vav3 or ΔCHVav3 (pCDH1-MCS1-EF1-copGFP). Cells were sorted for GFP+/YFP+ and treated with IODVA1 at the indicated concentrations. Cell cycle was analyzed at 18 h post BrdU incorporation.
SDS-PAGE, Pull-Down Assays and Immunoblotting:
Exponentially growing (6×106) p190-BCR-ABL Ba/F3 cells were treated with either vehicle or IODVA1 at indicated concentrations and time points. Active GTPase pulldown was done according to the manufacturer's instructions using GST-PAK1-GBD or GST-Rhotekin (Thermofisher, #16118 and #16116). Protein complexes were separated on SDS-PAGE and immunoblotted with anti-Rac1, anti-Cdc42 and anti-RhoA antibodies.
For analysis of expression, cells were lysed in RIPA buffer, supplemented with phosphatase and protease inhibitors, separated on SDS-PAGE, transferred on PVDF or nitrocellulose membrane and blotted with appropriate primary and secondary antibodies, as described previously (Lee et al., 2017; Chang et al., 2012). Relative signals were normalized to the unstimulated conditions after normalization to the total protein amount. Quantification was performed using Li-COR Image Studio.
Cloning:
Human Rac1 (GenBank accession no. NM_006908.4) was subcloned into pFastBacHTB vector (Invitrogen) and fused with an N-terminal His6-tag.
For pMalX(E)-based expression, full-length Vav3 was amplified with primers overlapping NotI restriction site of pMalX(E) vector with N-terminal linkers AAAA, AAAASEF or AAAASEFGS linkers. The finalized construct encoded MBPX(E)-linker-Vav3. For His6-MBP-N9-TEV-Vav3 construct, full-length Vav3 cDNA with a stop codon at the end of the coding sequence was amplified by PCR with primers overlapping pET28a-MBP-N9TEV-mOrange vector at the SspI site. The resulting construct encoded MBP-N9-Vav3-His6. All constructs were verified using Sanger sequencing using CCHCM DNA Core. To minimize aggregation and improve on quality of purified protein, the expression clones were tested with chaperone plasmids according to the manufacturer's protocol.
Protein Expression and Purification:
For membrane displacement assays, full-length human Rac1 was purified from baculovirus. pFastBacHTB-Rac1 was transformed into DH10 cells and the resulting bacmid was used to generate baculovirus in Sf9 cells. Rac1 was produced in TNAO38 insect cells and purified using Ni-IMAC chromatography (Zhang et al, 2014).
For production of recombinant Vav3, plasmids were co-transformed with chaperone plasmid Gro7 groEL-groES in BL21 (DE3) or T7 Express. Cultures were grown in LB, supplemented with metal mix (Studier 2005). Protein was purified using Ni-IMAC chromatography, dialyzed and further purified using size-exclusion gel filtration (HiLoad Superdex 200 16/60). Fractions were analyzed by SDS-PAGE and protein fractions containing Vav3 were pooled, concentrated to ˜10 mg/mL and flash frozen in liquid nitrogen. Final yields for MBP-N9-Vav3 and MBPXE-Vav3 were 5 mg and 20 mg per 6 L of culture, respectively.
Recombinant LARG (DH/PH) was purified as a MBP-fusion protein as described previously (Kristelly R. et al., 2003).
RhoGDI Extracting Prenylated Rac1 from Liposomes:
Displacement of prenylated-Rac1-GDP from synthetic liposomes by GST-RhoGDI1 in the presence and absence of IODVA1 was studied using liposome sedimentation assay as in Zhang et al., 2014. Briefly, liposomes were generated by using a defined composition of lipids (194 μg) containing 39% w/w phosphatidylethanolamine, 16% w/w phosphatidylcholine, 36% w/w phosphatidylserine, 4% sphingomyelin, and 5% w/w phosphatidylinositol 4,5-bisphosphate. Prenylated Rac1-GDP (1 μM) was added to liposomes suspended in protein buffer (20 mM Hepes, pH 7.4, 150 min NaCl, 5 min MgCl2, 3 min DTT) and incubated for 20 min on ice. GST-RHOGDI1 (2 μM) in the absence or presence of IODVA1 was added to the liposome/prenylated Rac1 and further incubated on ice for 30 min. The samples were then centrifuged at 20,000×g for 20 min at 4° C. Pellet and supernatant fractions were collected, separated on SDS-PAGE and immunoblotted for Rac1.
Microscale Thermophoresis (MST):
Purified Vav3, LARG or Rac (1 μM) were incubated with the indicated concentrations of IODVA1 at room temperature for 30 min. Samples were loaded into Zero Background MST Premium Coated capillaries and binding events were measured on Monolith NT.LabelFree (NanoTemper Technologies). Binding data were analyzed using Thermophoresis or Thermophoresis with Temperature Jump analysis as described previously (Jerabek-Willemsen et al., 2011). Data were normalized using fraction-bound binding. The 95% confidence interval for Kd values was 0.27 to 0.98 μM for Vav3, 5.9 to 10.37 μM for LARG, and 19.6 to 105.8 μM for Rac.
Stopped-Flow Spectrometry.
GTPase assay and nucleotide exchange reaction were performed with a Hi-Tech Scientific (SF-61) stopped-flow instrument as described (Nouri et al., 2016). The excitation wavelengths were 543 nm and 362 nm for tamraGTP and mantGppNHp, respectively. For GTPase assay, equal volumes (600 μl) of 0.2 μM Rac1-tamraGTP and 10 μM of p50GAP were used. GTPase assay as well the protein-protein interaction were performed in presence of 5% DMSO.
Animals and In Vivo Drug Administration:
Vav3-deficient mice (Fujikawa et al., 2003) and Rac1Δ/Δ+Rac2-deficient (Thomas et al., 2007) mice have been described previously. C57Bl/6 mice were commercially obtained (The Jackson Laboratory and Harlan Laboratories) and used as donors and/or recipients of transduction/transplantation models under a protocol approved by The Cincinnati Children's Hospital Medical Center Institutional Animal Care and Use Committee. For in vivo drug administration, Alzet implantable osmotic pumps (Model 2002, Durect) were used according to the manufacturer's protocol and implantation was done as described previously (Thomas et al., 2007).
Histology:
Embedded tissues were cut into 4 μm sections then immunohistochemically stained using the Mouse on Mouse kit (MoM kit, Vector Laboratories). Tissue sections were subjected to sodium citrate antigen retrieval, pretreated with 0.3% hydrogen peroxide, blocked according to kit directions, and incubated with an antibody to phosphorylated Vav3 (1:200 dilution, Abcam) and HRP-conjugated anti-rabbit secondary (Vector Laboratories). Staining was completed with the DAB Peroxidase kit (Vector Laboratories) and counterstained with hematoxylin. Tissue sections were coverslipped with Cytoseal 60 and images acquired with a Nikon Eclipse Ci microscope.
CFU-proB Assay:
B-cell lineage colony-forming units (CFU-proB) were quantified post 9-day culture of leukemic BM cells or sorted for p190-BCR-ABL-expressing B-cell progenitors in M3134 methylcellulose (StemCell Technologies) supplemented with 30% FBS (for mouse B lymphoid colony forming cells; StemCell Technologies), 2 min L-glutamine (Invitrogen), 1% penicillin-streptomycin (Invitrogen), 100 μM β-mercaptoethanol (Fisher-Scientific), 1% BSA (Sigma-Aldrich), 20 ng/mL of recombinant mouse IL-7 (PeproTech), and 100 ng/mL of recombinant mouse SCF (PeproTech).
Cell Cycle Analysis:
Cell cycle was analyzed by using in vitro incorporation of 5-Bromo-2-deoxyuridine using Brdu Flow kit (BD Pharmingen, Cat #552598). Briefly, mice leukemic progenitors were incubated with 1 min BrdU solution for 45 minutes, cells were further fixed and permeabilized. DNAse treatment was done according to the instructions and stained with anti BrdU and apoptosis was analyzed by 7-AAD staining through flow cytometry analysis.
Flow Cytometry Analysis:
Red blood cells were removed from the peripheral blood samples using fixative-free lysis buffer (BD Pharm Lyse lysing buffer Cat #555899). After a single wash in PBS, cells were stained with anti-B220 APC-Cy7 antibody. Stained cells were washed once and analyzed by flow cytometry.
Primary PDX Ex Vivo Drug Treatments:
Primary patient specimens were obtained from patients at CCHMC according to Institutional Review Board Approved protocols (#2008-0021 and #2008-0658). Samples were subjected to RBC lysis and the isolated WBCs were mixed with OKT3 anti-CD3 antibody to eliminate the potential for xenogenic Graft Versus Host Disease before injection into busulfan conditioned NSG or NRG mice10. Spleen preparations from mice successfully engrafted with B-ALL were co-cultured with MS-5 or OP9 stroma in MEMα media supplemented with 20% FBS and 10 ng/mL recombinant human SCF (Kit-L), Flt3L, and IL-7 (KF7). IODVA1 was added 24 h after initial seeding. Co-cultures were collected by trypsinization after 7 days and cell counts were performed with trypan blue. Flow cytometry was performed with mCD45-APC-Cy7 (BD), hCD45-FITC (BD), hCD19-VioBlue (Miltenyi Biotech), and 7-AAd (for viability) to determine percentage of human ALL in the cultures. Total absolute ALL cell numbers were determined by multiplying cell counts by percentage human ALL cells.
NIRA2 is a Potent Inhibitor of p190-BCR-ABL Expressing Cells in Cellular Assays.
The efficacy of NIRA2 was tested in Ba/F3 cells transduced with p190-BCR-ABL, a commonly used Ph+ B-ALL cell model or with empty vector Mieg3. Ba/F3 cells are bone marrow-derived, interleukin-3 dependent, murine pro-B cells widely used in studying the mechanism of leukemia initiation and progression and targeted-therapy discovery. Cells were grown in suspension in the presence of IL-3 and NIRA2 (0 to 300 nM) and counted cells daily for 3 days by trypan blue exclusion (
NIRA2 is not a Kinase Inhibitor.
To test if NIRA2 is a kinase inhibitor, its potential to interfere with the ability of 485 recombinant wild-type and mutant kinases to hydrolyze ATP was evaluated. Each kinase was tested twice at one NIRA2 concentration of 0.5 μM in the presence of ATP (at either the Km concentration or at 10 μM) and Mg2+ (5 mM) and data were averaged and compared to vehicle DMSO control. A plot of the replicates compared to vehicle control set at 0% is shown in
NIRA2 Inhibits Tumor Growth of a Colon Cancer Mouse Model.
The in vivo efficacy of NIRA2 was tested against a xenograft mouse model generated with the murine colon adenocarcinoma MC38 cells. Cells were injected subcutaneously in the flank of a hind leg. Each injection contained one million cells of Matrigel+MC38 in a volume of 100 pt. Tumors were calipered until an average size of 100-200 mm3 is reached. Animals were sorted in into treatment groups (N=10) and administered (5× a week) by IP 250 μL of a PBS solution containing vehicle control, IODVA1 (1 mM), or NIRA2 (0.5 mM). Treatments lasted 2 weeks Animals were euthanized as tumor size reached 2 cm3 or at the end of the study. Tumors were measured daily and mice weighed 3 times a week. As shown in
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All percentages and ratios are calculated by weight unless otherwise indicated.
All percentages and ratios are calculated based on the total composition unless otherwise indicated.
It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.”
Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.
While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims
1. A composition comprising
- and a pharmaceutically acceptable carrier; wherein A=NH, NR8, S, O, C═C, N═C, C═N wherein R1, R2 are independently substituted or unsubstituted* aryl or heteroaryl rings wherein R3=singly or multiply substituted as H, D, Halo, CN, C1-C4 Alkyl, C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 Alkyl amino, or C1-C4 mercapto wherein R8=H, M;
- and all tautomers thereof.
2. The composition of claim 1, wherein said compound has the structure: and all tautomers thereof.
3. The composition of claim 1, wherein said compound has the structure: and all tautomers thereof.
4. The composition of claim 1, wherein said compound has the structure: and all tautomers thereof, and a pharmaceutically acceptable carrier.
5. The composition of claim 1, wherein said compound has the structure: and all tautomers thereof, and a pharmaceutically acceptable carrier.
6. A composition comprising
- wherein R1, R2, and R4 are independently selected from H, D, Halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et, or Pr, and all tautomers thereof;
- wherein R3 is singly or multiply substituted as H, D, Halo, CN, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylsufonyl; C1-C4 alkylamino, or C1-C4 mercapto;
- wherein A is selected from NH, NRB, S, O, C═C, N═C, C═N, wherein R8 is H or Me; wherein each Q is independently selected from N, C, and S;
- and a pharmaceutically acceptable carrier.
7. The composition of claim 6, wherein said compound has the structure: or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier
8. The composition of claim 6, wherein said compound has the structure: or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
9. A method of treating cancer in an individual in need thereof, comprising the step of administering to said individual a compound of claim 1.
10. The method of claim 9, wherein said cancer is selected from a leukemia, chemotherapy-resistant leukemia, immunotherapy-resistant leukemia, relapsed leukemia, and other targeted-therapy resistant leukemias.
11. The method of claim 9, wherein said cancer is selected from adenocarcinoma, breast cancer, prostate cancer, ovarian cancer, endometrial cancer, thyroid cancer, lung cancer, colorectal cancer, pancreatic cancer, and cervical cancer.
12. The method of claim 9, wherein said cancer is a Ras-driven cancer, including RASopathies.
13. The method of claim 9, comprising the steps of:
- a. determining the level of Vav3 or of the Rac GTPase in a biopsy obtained from a cancer in an individual; and
- b. administering a compound of claim 1 to said individual where the level of Vav3 or of the Rac GTPase is elevated as compared to a control.
14. A method of treating cancer in an individual in need thereof, comprising the step of administering to said individual a compound of claim 6.
15. The method of claim 14, wherein said cancer is selected from a leukemia, chemotherapy-resistant leukemia, immunotherapy-resistant leukemia, relapsed leukemia, and other targeted-therapy resistant leukemias.
16. The method of claim 14, wherein said cancer is selected from adenocarcinoma, breast cancer, prostate cancer, ovarian cancer, endometrial cancer, thyroid cancer, lung cancer, colorectal cancer, pancreatic cancer, and cervical cancer.
17. The method of claim 14, wherein said cancer is a Ras-driven cancer, including RASopathies.
18. The method of claim 14, comprising the steps of:
- a. determining the level of Vav3 or of the Rac GTPase in a biopsy obtained from a cancer in an individual; and
- b. administering a compound of claim 6 to said individual where the level of Vav3 or of the Rac GTPase is elevated as compared to a control.
Type: Application
Filed: May 1, 2020
Publication Date: Nov 5, 2020
Patent Grant number: 11564913
Inventors: Nicolas Nassar (Cincinnati, OH), William Seibel (Liberty Township, OH), Anjelika Gasilina (Bethesda, MD), Jose Cancelas (Cincinnati, OH)
Application Number: 16/864,961
